Methods for treating atherosclerosis

ABSTRACT

The invention provides compounds, pharmaceutical compositions and methods for treating atherosclerosis, inflammation, thrombosis and other conditions and for decreasing or prevention of accumulation of cholesterol in a subject by modifying LCAT polypeptide.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a continuation of U.S. Nonprovisional applicationSer. No. 13/087,261, filed Apr. 14, 2011, which is a division of U.S.Nonprovisional application Ser. No. 11/823,251, filed Jun. 26, 2007,which claims the benefit of priority of U.S. Provisional PatentApplication No. 60/816,415, filed on Jun. 26, 2006, all of which arehereby incorporated by reference.

REFERENCE TO SEQUENCE LISTING

The present application incorporates by reference in its entirety allsubject matter contained in the attached sequence listing which is in.txt format and is identified by the name of the file, Seqlist.txt,created on Mar. 26, 2013, the size of which file is 20 KB.

FIELD OF THE INVENTION

This invention relates generally to the field of medicine and, morespecifically, to compositions and methods for treating coronary heartdisease and atherosclerosis.

BACKGROUND OF THE INVENTION

Over 50 million Americans have cardiovascular problems, and many othercountries face high and increasing rates of cardiovascular disease. Itis the number one cause of death and disability in the United States andmost European countries. By the time that heart problems are detected,the underlying cause, atherosclerosis, is usually quite advanced, havingprogressed for decades.

Atherosclerosis is a polygenic complex disease of mammals characterizedby the deposits or plaques of lipids and other blood derivatives in thearterial walls (aorta, coronary arteries, carotid). These plaques can becalcified to a greater or lesser extent according to the progression ofthe process. They are also associated with the accumulation of fattydeposits consisting mainly of cholesterol esters in the arteries.Cholesterol accumulates in the foam cells of the arterial wall, therebynarrowing the lumen and decreasing the flow of blood. This isaccompanied by a thickening of the arterial wall, with hypertrophy ofthe smooth muscle, the appearance of foam cells and the accumulation ofthe fibrous tissue. Hypercholesterolemia can therefore result in veryserious cardiovascular pathologies such as infarction, peripheralvascular disease, stroke, sudden death, cardiac decompensation, cerebralvascular accidents and the like.

The cholesterol is carried in the blood by various lipoproteinsincluding the low-density lipoproteins (LDL) and the high-densitylipoproteins (HDL). The LDL is synthesized in the liver and makes itpossible to supply the peripheral tissues with cholesterol. In contrast,the HDL captures cholesterol molecules from the peripheral tissues andtransports them to the liver where they are converted to bile acids andexcreted. The development of atherosclerosis and the risk of coronaryheart disease (CHD) inversely correlate to the levels of HDL in theserum. Gordon et al. (1989) N. Engl. J. Med. 321: 1311; Goldbourt et al.(1997) Thromb Vasc. Biol. 17: 107. Low HDL cholesterols often occur inthe context of central obesity, diabetes and other features of themetabolic syndrome. Goldbourt et al., supra. It has been suggested thatlow HDL cholesterol levels are associated with an increased risk of CHD,while high concentrations of HDL have a protective effect against thedevelopment of premature atherosclerosis. Gordon et al. (1986)Circulation 74: 1217. Studies demonstrated that the risk for developingclinical atherosclerosis in men drops 3% with a 1% increase in theconcentration of HDL in plasma. Gordon et al. (1989) N. Engl. J. Med.321: 1311. It has been established that concentrations of LDLcholesterol can be reduced by treatment with statins, inhibitors of thecholesterols biosynthesis enzyme 3-hydroxyl-3-methylglutary Coenzyme Areductase and thereby this treatment has been used as a successfulapproach for reducing the risk for atherosclerosis where the primaryindication is high LDL level. However, it remains unclear whetherstatins are beneficial for patients whose primary lipid abnormality islow HDL cholesterol.

Lecithin-cholesterol acyltransferase (LCAT) is an enzyme which catalyzesthe esterification of free cholesterol by the transfer of an acyl groupfrom phosphatidylcholine onto 3-hydroxyl group of the cholesterol,forming cholesteryl ester and lysophosphatidylcholine. McLean et al.(1986) Proc. Natl. Acad. Sci. 83: 2335 and McLean et al. (1986) NucleicAcids Res. 14(23): 9397. LCAT is synthesized in the liver and releasedinto the plasma, where it is combined with HDL, called anti-atherogeniclipoproteins. These HDL particles have the capacity to accept the excesscholesterol, which is then esterified by LCAT. The cholesteryl estermolecules in the HDL particles are either transported to the liverdirectly through SR-BI receptor, or transferred to apoB-containinglipoproteins, including very low density lipoproteins (VLDL) and LDL,mediated by CETP, and then transported to the liver through LDL-receptorpathway. This mechanism, called reverse cholesterol transport (Glomset(1968) J. Lipid Res. 9:155), allows the removal of excess cholesterolfrom the body, and therefore is involved in the prevention ofatherogenesis. LCAT plays a significant role in this process by creatinga gradient of free cholesterol between the plasma membranes and thecirculating lipoproteins.

This invention provides compositions comprising LCAT modified toincrease enzymatic activity and/or stability and methods for treatmentand prevention of atherosclerosis, CHD, and other conditions, includinginflammation, thrombosis, and disorders associated with these conditionsusing the compounds and compositions of the invention.

SUMMARY OF THE INVENTION

The present invention provides methods for treating atherosclerosis in asubject in need thereof comprising administering to the subject atherapeutically effective amount of a compound of Formula I

wherein all substituents are as indicated in Detailed Description below,or a pharmaceutically acceptable salts thereof. In one aspect, theinvention provides methods for treating atherosclerosis in a subjectwherein X and Y are each —N═. In another aspect, Z can be —S—. In afurther aspect, L can be —S—. In one aspect, R¹ can be CN. In anotheraspect, R² can be SR³. In one aspect, R³ can be C₁-C₄ alkyl, forexample, methyl.

In one aspect, the invention provides methods for treatingatherosclerosis, inflammation, thrombosis, and conditions associatedwith these disorders in a subject comprising administering to thesubject in need thereof a therapeutically effective amount of a compoundselected from the group consisting of3-(5-(methylthio)-1,3,4-thiadiazol-2-ylthio)pyrazine-2-carbonitrile,3-(5-(ethylthio)-1,3,4-thiadiazol-2-ylthio)pyrazine-2-carbonitrile,3-(5-(allylthio)-1,3,4-thiadiazol-2-ylthio)pyrazine-2-carbonitrile,3-(5-(propylthio)-1,3,4-thiadiazol-2-ylthio)pyrazine-2-carbonitrile,3-(5-(butylthio)-1,3,4-thiadiazol-2-ylthio)pyrazine-2-carbonitrile,3-(5-(isobutylthio)-1,3,4-thiadiazol-2-ylthio)pyrazine-2-carbonitrile,3-(5-(pentylthio)-1,3,4-thiadiazol-2-ylthio)pyrazine-2-carbonitrile,3-(5-(dodecylthio)-1,3,4-thiadiazol-2-ylthio)pyrazine-2-carbonitrile,3-(5-(benzylthio)-1,3,4-thiadiazol-2-ylthio)pyrazine-2-carbonitrile,3-(5-mercapto-1,3,4-thiadiazol-2-ylthio)pyrazine-2-carbonitrile,3-(5-(isopropylthio)-4-methyl-4H-1,2,4-triazol-3-ylthio)pyrazine-2-carbonitrile,3-(5-(methylthio)-1,2,4-thiadiazol-3-ylthio)pyrazine-2-carbonitrile,3-(5-methyl-1,3,4-thiadiazol-2-ylthio)pyrazine-2-carbonitrile,3-(5-butyl-1,3,4-thiadiazol-2-ylthio)pyrazine-2-carbonitrile,3-(4-methyl-4H-1,2,4-triazol-3-ylthio)pyrazine-2-carbonitrile,3-(1-methyl-1H-imidazol-2-ylthio)pyrazine-2-carbonitrile, and2-chloro-3-(5-(methylthio)-1,3,4-thiadiazol-2-ylthio)pyrazine or apharmaceutically acceptable salt thereof.

The invention further provides methods for treating atherosclerosis,inflammation, thrombosis, and conditions associated with these disordersin a subject comprising administering to the subject in need thereof atherapeutically effective amount of a modified LCAT comprising areplacement of the amino acid residue 31 by a cysteine residue, whereinthe cysteine residue is modified by replacing the thiol hydrogen with3-pyrazinyl-2-carbonitrile.

In one aspect, the modified LCAT can be administered intravenously, forexample, by bolus.

The invention provides methods for treating an LCAT-mediated diseasecomprising administering to a subject in need thereof an effectiveamount of a modified LCAT comprising a replacement of the amino acidresidue 31 by a cysteine residue, wherein the cysteine residue ismodified by replacing the thiol hydrogen with3-pyrazinyl-2-carbonitrile. In one aspect, the LCAT-mediated disease canbe atherosclerosis, thrombosis, coronary heart disease, high bloodpressure, LCAT deficiency syndrome, Alzheimer's disease, cornealopacity, metabolic syndrome, dyslipidemia, myocardial infarction,stroke, critical limb ischemia or angina, inflammation, and conditionsassociated with these disorders.

The invention further provides methods for increasing HDL cholesterol ina subject comprising administering to the subject in need thereof atherapeutically effective amount of a modified LCAT comprising areplacement of the amino acid residue 31 by a cysteine residue, whereinthe cysteine residue is modified by replacing the thiol hydrogen with3-pyrazinyl-2-carbonitrile. In one aspect, the invention providesmethods for preventing accumulation of cholesterol in a subjectcomprising administering to the subject a pharmaceutical compositioncomprising a therapeutically effective amount of a modified LCATcomprising a replacement of the amino acid residue 31 by a cysteineresidue, wherein the cysteine residue is modified by replacing the thiolhydrogen with 3-pyrazinyl-2-carbonitrile, and a pharmaceuticallyacceptable carrier or excipient.

The invention provides a pharmaceutical composition comprising amodified LCAT comprising a replacement of the amino acid residue 31 by acysteine residue, wherein the cysteine residue is modified by replacingthe thiol hydrogen with 3-pyrazinyl-2-carbonitrile, and apharmaceutically acceptable carrier.

In one aspect, the subject can be mammalian. In another aspect, thesubject can be human.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically represents sequences of human (A, SEQ ID NO:1),mouse (B, SEQ ID NO:2), rat (C, SEQ ID NO:3), and consensus LCATpolypeptide (D, SEQ ID NO:4).

FIG. 2A demonstrates activity and specificity of the compounds of theinvention on LCAT enzyme.

FIG. 2B illustrates the mechanism of action of compounds of theinvention on LCAT enzyme.

FIG. 3 summarizes data showing that compounds of the invention increaseLCAT enzyme activity in a dose dependent manner in BALB/c mice.

FIG. 4 demonstrates that treatment with the compounds of the inventionincreases HDL cholesterol levels in CD1 mice.

FIG. 5 illustrates the time course of LCAT activation and the levels ofHDL in mice following a single doze of the compounds of the invention.

FIG. 6 demonstrates that treatment with the compounds of the inventionincreases HDL levels (A) and decreases apoB-containing lipoprotein (C)in vivo. LpB cholesterol contents were obtained by subtracting HDL fromTC (B).

FIG. 7 provides elution profiles indicating that treatment with thecompounds of the invention increases HDL-Ch levels (A), increases HDLparticle size, and decrease TG levels in the VLDL fraction (B) in vivo.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

The term “pharmacologically active” means that a substance so describedis determined to have activity that affects a medical parameter ordisease state.

“Substantially homogenous” as used herein with reference to an LCATpreparation means that the preparation includes a single species of atherapeutic LCAT compound detectable in the preparation of totaltherapeutic molecules in the preparation, unless otherwise stated at aspecific percentage of total therapeutic molecules. In general, asubstantially homogenous preparation is homogenous enough to display theadvantages of a homogenous preparation, e.g., ease in clinicalapplication in predictability of lot to lot pharmacokinetics.

“Bioefficacy” refers to the capacity to produce a desired biologicaleffect. Bioefficacy of different compounds, or different dosages of thesame compound, or different administrations of the same compound aregenerally normalized to the amount of compound(s) to permit appropriatecomparison.

The term “LCAT” or “lecithin-cholesterol acyltransferase”, as usedherein, refers to a glycoprotein enzyme that catalyzes the synthesis ofcholesterol esters and lysolecithin from phosphatidylcholine andunesterified cholesterol present in lipoproteins. This enzyme isproduced primarily by the liver and circulates in blood reversibly boundto lipoproteins. Human LCAT has a polypeptide mass of 49 kDa, or around67 kDa with added carbohydrate mass. Any polypeptide variants orfragments of mammalian LCAT that have the LCAT enzymatic activity asdescribed above and in more detail below are useful as compounds and inmethods of the instant invention. A polypeptide fragment is a stretch ofamino acid residues of at least 12 contiguous amino acids from aparticular sequence. Some mammalian LCAT sequences for obtaining themodified LCAT useful in this invention are represented in FIG. 1.

The term “modified LCAT,” “derivatized LCAT,” or “LCAT derivative”refers to lecithin-cholesterol acyltransferase as defined above, witheither increased enzymatic activity, wherein the enzymatic activity ofthe modified LCAT is increased compared to the wild type LCAT asmeasured in the same assay conditions; or increased plasma stability orhalf-life time, wherein LCAT stability is improved compared to wild typeLCAT plasma stability as measured in the same assay conditions. Assaysfor measuring LCAT enzyme activity include, e.g., use of apoAI-liposomeassay and use of plasma LCAT activity assay, which determine cholesterolesterification rate in an artificial system and in a physiologicallyrelevant system, respectively. Assays for measuring LCAT stability invivo includes ELISA, which determines the half-life of recombinant LCATprotein in the blood after LCAT protein administration.

“Atherosclerosis” refers to a condition characterized by the hardeningand/or narrowing of the arteries caused by the buildup of athermatousplaque inside the arterial walls. The atheromatous plaque is divided inthree components, (1) the atheroma, a nodular accumulation of a softflaky material at the center of large plaques, composed of macrophagesnearest the lumen of the artery; (2) underlying areas of cholesterolcrystals; (3) calcification at the outer base of more advanced lesions.Indicators of atherosclerosis include, for example, the development ofplaques in the arteries, their calcification, the extent of which can bedetermined by Sudan IV staining, or the development of foam cells inarteries. The narrowing of the arteries can be determined by coronaryangioplasty, ultrafast CT, or ultrasound.

“Inflammation” or “inflammatory disorder” refers to a localized,protective response elicited by injury or destruction of tissues, whichserves to destroy, dilute or wall off (sequester) both the injuriousagent and the injured tissue. The term “inflammatory disease” or“inflammatory condition” as used herein, means any disease in which anexcessive or unregulated inflammatory response leads to excessiveinflammatory symptoms, host tissue damage, or loss of tissue function.Additionally, the term “autoimmune disease,” as used herein, means anygroup of disorders in which tissue injury is associated with humoral orcell-mediated responses to the body's own constituents. The term“allergic disease,” as used herein, means any symptoms, tissue damage,or loss of tissue function resulting from allergy. The term “arthriticdisease,” as used herein, means any of a large family of diseases thatare characterized by inflammatory lesions of the joints attributable toa variety of etiologies. The term “dermatitis,” as used herein, meansany of a large family of diseases of the skin that are characterized byinflammation of the skin attributable to a variety of etiologies. Theterm “transplant rejection,” as used herein, means any immune reactiondirected against grafted tissue (including organ and cell (e.g., bonemarrow)), characterized by a loss of function of the grafted andsurrounding tissues, pain, swelling, leukocytosis and thrombocytopenia.

“Thrombosis” and “thrombosis-related disorder” refer to abnormalthrombus formation that causes obstruction of blood vessels andconditions associated with such obstruction. Blood vessels operate undersignificant shear stresses that are a function of blood flow shear rate.Frequently, there is damage to small blood vessels and capillaries. Whenthese vessels are damaged, hemostasis is triggered to stop the bleeding.Under typical circumstances, such an injury is dealt with through asequence of events commonly referred to as the “thrombus formation”.Thrombus formation is dependent upon platelet adhesion, activation andaggregation and the coagulation cascade that culminates in theconversion of soluble fibrinogen to insoluble fibrin clot. Thrombusformation at site of wound prevents extravasation of blood components.Subsequently, wound healing and clot dissolution occurs and blood vesselintegrity and flow is restored.

The term “HDL” refers to the high-density lipoproteins.

The term “LDL”, as used herein, means the low-density lipoproteins.

The term “VLDL” refers to the very low density lipoproteins.

The term “treatment” or “treating” includes the administration, to asubject in need, of an amount of a compound of the invention which willinhibit, decrease or reverse development of, for example, a pathologicalatherosclerosis, inflammatory, or thrombosis-related condition asdisclosed herein without limitation. In another aspect, treatment asused herein means the administration, to a subject in need, of an amountof a compound of the invention, which will increase HDL cholesterollevels. “Inhibiting,” in connection with inhibiting atherosclerosis, isintended to mean preventing, retarding, stabilizing, or reversingformation or growth of atheromatous plaques, inflammatory condition, orthrombosis-related indication. Treatment of diseases and disordersherein is intended to also include therapeutic administration of acompound of the invention (or a pharmaceutical salt, derivative orprodrug thereof) or a pharmaceutical composition containing the compoundto a subject (i.e., an animal, for example a mammal, such as a human)believed to be in need of treatment for diseases and disorders, such as,for example, inflammation, thrombosis, coronary heart disease, highblood pressure, LCAT deficiency syndrome, Alzheimer's disease, cornealopacity, metabolic syndrome, dyslipidemia, myocardial infarction,stroke, critical limb ischemia, angina and the like. Treatment alsoencompasses administration of the compound or pharmaceutical compositionto subjects not having been diagnosed as having a need thereof, i.e.,prophylactic administration to the subject, such as prevention ofaccumulation of cholesterol. Generally, the subject is initiallydiagnosed by a licensed physician and/or authorized medicalpractitioner, and a regimen for prophylactic and/or therapeutictreatment via administration of the compound(s) or compositions of theinvention is suggested, recommended or prescribed.

The phrase “therapeutically effective amount” is the amount of thecompound of the invention that will achieve the goal of improvement indisorder severity and the frequency of incidence. The improvement indisorder severity includes, for example, prevention of accumulation ofcholesterol in vessel walls increasing of blood levels of HDLcholesterol, the reversal of atherosclerosis, as well as slowing downthe progression of atherosclerosis, prevention or treatment ofinflammatory disorders, and prevention or treatment ofthrombosis-relating conditions.

As used herein, the term “subject” is intended to mean a human or othermammal, exhibiting, or at risk of developing, atherosclerosis, aninflammatory condition or thrombosis. Such an individual can have, or beat risk of developing, for example, atherosclerosis associated withconditions such as thrombosis, coronary heart disease, high bloodpressure, LCAT deficiency syndrome, Alzheimer's disease, cornealopacity, metabolic syndrome, dyslipidemia, myocardial infarction,stroke, critical limb ischemia, angina and the like. The prognostic andclinical indications of these conditions are known in the art.

II. LCAT Compounds

The invention provides compounds, pharmaceutical compositions andmethods for treating atherosclerosis and for decrease or prevention ofaccumulation of cholesterol in a subject by modifying LCAT polypeptide.In one aspect, the modified LCAT can be obtained by activation usingsmall molecule compounds of the invention. In another aspect, modifiedLCAT can be obtained by modification at amino acid residue 31, forexample, via the covalent binding to 3-pyrazinyl-2-carbonitrile.

Assays for LCAT activity, plasma stability (enzyme half-life in theplasma) or the plasma LCAT protein levels are known in the art. AbsoluteLCAT activity in the serum and endogenous cholesterol esterificationrate can be determined as described, e.g., in Albers J. et al. (1986)Methods in Enzymol. 129: 763-783; Dobiasova M. et al. (1983) Adv. LipidRes. 20: 107-194. In one aspect, LCAT activity can be determined bymeasuring the conversion of radiolabeled cholesterol to cholesterolester after incubation of LCAT and radiolabeled LCAT substratescontaining Apo A-I. Cholesterol esterification rate (nmol CE/mL perhour) can be measured by determining the rate of conversion of labeledcholesterol to cholesteryl ester after incubation of plasma that isradiolabeled with a trace amount of radioactive cholesterol byequilibration with a [¹⁴C] cholesterol-albumin mixture at 4° C. Theendogenous cholesterol esterification rate (as determined with plasmaLCAT activity assay) reflects not only on mass of LCAT, but also thenature and amount of LCAT substrate and cofactor present in the serum,and therefore provides a better measure of the therapeutic LCATactivity.

Assays for measuring LCAT stability (half-life) in the blood and plasmaLCAT protein concentration are also known in art. After administration,recombinant LCAT protein levels in the plasma can be determined by usingELISA described by JR Crowther (ELISA theory and practice, methods inmolecular Biology Volume 42). Reagents for measuring LCAT stability andprotein concentration include anti-LCAT antibodies, which arecommercially available from several vendors. Examples of use of thisassay to determine activity and/or stability of the modified LCAT aregiven below.

Compounds of the Invention

Generally, reference to a certain element such as hydrogen or H is meantto include all isotopes of that element. For example, if an R group isdefined to include hydrogen or H, it also includes deuterium andtritium. Compounds comprising radioisotopes such as tritium, ¹⁴C, ³²Pand ³⁵S are thus within the scope of the invention. Procedures forinserting such labels into the compounds of the invention will bereadily apparent to those skilled in the art based on the disclosureherein.

In general, “substituted” as used herein refers to a group, such asthose defined below, in which one or more bonds to a hydrogen atomcontained therein are replaced by a bond to non-hydrogen or non-carbonatoms such as, but not limited to, a halogen atom such as F, Cl, Br, andI; an oxygen atom in groups such as hydroxyl groups, a nitrogen atom ingroups such as amines, amides, alkylamines, dialkylamines, arylamines,alkylarylamines, diarylamines, N-oxides.

Substituents, including alkyl and ring groups, may be either monovalentor polyvalent depending on the context of their usage. For example, ifdescription contained the group R¹—R²—R³ and R² was defined as C₁₋₆alkyl, then the R² alkyl would be considered polyvalent because it mustbe bonded to at least R¹ and R³. Alternatively, if R¹ was defined asC₁₋₆ alkyl, then the R¹ alkyl would be monovalent (excepting any furthersubstitution language).

In general, “unsubstituted” as used herein with reference to a group,means that the group does not have one or more bonds to a hydrogen orcarbon atom contained therein replaced by a bond to non-hydrogen ornon-carbon atom, as described above.

In general, “alkyl” as used herein either alone or within other termssuch as “haloalkyl”, “alkylamino” and “cycloalkyl”, refers to linear,branched or cyclic radicals having one to about twelve carbon atoms.“Cycloalkyl” is also used exclusively herein to refer specifically tofully or partially saturated cyclic alkyl radicals. Examples of “alkyl”radicals include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,sec-butyl, tert-butyl, pentyl, isoamyl, hexyl, cyclopropyl, cyclopentyl,cyclohexyl and the like.

In general, “C_(a-b) alkyl” as used herein refers to an alkyl groupcomprising from a to b carbon atoms in a branched, cyclical or linearrelationship or any combination of the three. The alkyl groups describedin this section may also contain double or triple bonds. Examples ofC₁₋₈ alkyl include, but are not limited to the following:

In general, “halogen” and “halo” as used herein, refers to a halogenatoms selected from F, Cl, Br and I.

In general, “haloalkyl”, as used herein refers to radicals wherein anyone or more of the alkyl carbon atoms is substituted with halo asdefined above. Specifically embraced are monohaloalkyl, dihaloalkyl andpolyhaloalkyl radicals including perhaloalkyl. A monohaloalkyl radical,for example, may have either an iodo, bromo, chloro or fluoro atomwithin the radical. Dihalo and polyhaloalkyl radicals may have two ormore of the same halo atoms or a combination of different halo radicals.Examples of haloalkyl radicals include fluoromethyl, difluoromethyl,trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl,pentafluoroethyl, heptafluoropropyl, difluorochloromethyl,dichlorofluoromethyl, difluoroethyl, difluoropropyl, dichloroethyl anddichloropropyl.

In general, “C_(a-b) haloalkyl” as used herein refers to an alkyl group,as described above, wherein any number, but at least on of the hydrogenatoms attached to the alkyl chain are replaced by F, Cl, Br or I.Examples of haloalkyl includes, without limitation, trifluoromethyl,pentafluoroethyl and the like.

In general, “heteroalkyl” as used herein refers to an alkyl having oneor more of the carbon atoms replaced by a heteroatom, selected fromnitrogen, oxygen and sulfur. For example, a heteroalkyl would include anether or a thioether chain, or an alkoxide moiety, wherein theheteroatom is in the linear region of the moeity. The term also includesmoieties where the heteroatom is in a branched region. For example, theterm includes 2-amino-n-hexane or 5-hydroxy-pentane.

In general, “hydroxyalkyl” as used herein refers to linear or branchedalkyl radicals having one to about ten carbon atoms any one of which maybe substituted with one or more hydroxyl radicals. Examples of suchradicals include hydroxymethyl, hydroxyethyl, hydroxypropyl,hydroxybutyl and hydroxyhexyl.

In general, “alkoxy” as used herein refers to linear or branchedoxy-containing radicals each having alkyl portions of one to about tencarbon atoms. Examples of such radicals include methoxy, ethoxy,propoxy, butoxy and tert-butoxy. Alkoxy radicals may be furthersubstituted with one or more halo atoms, such as fluoro, chloro orbromo, to provide “haloalkoxy” radicals. Examples of lower haloalkoxyradicals having one to three carbon atoms include fluoromethoxy,chloromethoxy, trifluoromethoxy, trifluoroethoxy, fluoroethoxy andfluoropropoxy.

In general, “sulfonyl”, as used herein whether alone or linked to otherterms such as alkylsulfonyl, refers to divalent radicals —SO₂—.

In general, “aryl”, as used herein alone or in combination, refers to acarbocyclic aromatic system containing one, two or three rings whereinsuch rings may be attached together in a fused manner. The term “aryl”includes, without limitation, aromatic radicals such as phenyl,naphthyl, indenyl, tetrahydronaphthyl, and indanyl. The “aryl” group mayhave 1 to 3 substituents such as alkyl, hydroxyl, halo, haloalkyl,nitro, cyano, alkoxy and alkylamino. “Aryl” also includes the moietywherein the aromatic carbocycle is fused with a C₃₋₆ cycloalkyl bridge,wherein the bridge optionally includes 1, 2 or 3 heteroatoms selectedfrom N, O and S. For example, phenyl substituted with —O—CH₂—O— formsthe aryl benzodioxolyl substituent.

“Saturated or unsaturated” means a substitutent that is completelysaturated, completely unsaturated, or has any degree of unsaturation inbetween. Examples of a saturated or unsaturated 6-membered ringcarbocycle would include phenyl, cyclohexyl, cyclohexenyl andcyclohexadienyl.

In general, “salt” refers to a salt form of a free base compound of thepresent invention, as appreciated by persons of ordinary skill in theart. Salts may be prepared by conventional means, known to those skilledin the art. In general, “pharmaceutically-acceptable”, when used inreference to a salt, refers to salt forms of a given compound, which arewithin governmental regulatory safety guidelines for ingestion and/oradministration to a subject. The term “pharmaceutically-acceptablesalts” embraces salts commonly used to form alkali metal salts and toform addition salts of free acids or free bases. The nature of the saltis not critical, provided that it is pharmaceutically acceptable. Somespecific examples are acetate; trifluoroacetate; hydrohalides, such ashydrochloride and hydrobromide; sulfate; citrate; tartrate; glycolate;and oxalate.

Suitable pharmaceutically-acceptable acid addition salts of compounds ofFormula I may be prepared from an inorganic acid or from an organicacid. Examples of such inorganic acids are hydrochloric, hydrobromic,hydroiodic, nitric, carbonic, sulfuric and phosphoric acid. Appropriateorganic acids may be selected from aliphatic, cycloaliphatic, aromatic,arylaliphatic, heterocyclic, carboxylic and sulfonic classes of organicacids, example of which are formic, acetic, adipic, butyric, propionic,succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic,glucuronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic,anthranilic, mesylic, 4-hydroxybenzoic, phenylacetic, mandelic, embonic(pamoic), methanesulfonic, ethanesulfonic, ethanedisulfonic,benzenesulfonic, pantothenic, 2-hydroxyethanesulfonic, toluenesulfonic,sulfanilic, cyclohexylaminosulfonic, camphoric, camphorsulfonic,digluconic, cyclopentanepropionic, dodecylsulfonic, glucoheptanoic,glycerophosphonic, heptanoic, hexanoic, 2-hydroxy-ethanesulfonic,nicotinic, 2-naphthalenesulfonic, oxalic, palmoic, pectinic,persulfuric, 2-phenylpropionic, picric, pivalic propionic, succinic,tartaric, thiocyanic, mesylic, undecanoic, stearic, algenic,(3-hydroxybutyric, salicylic, galactaric and galacturonic acid.

Suitable pharmaceutically acceptable base addition salts of compounds ofFormula I include metallic salts, such as salts made from aluminum,calcium, lithium, magnesium, potassium, sodium and zinc, or salts madefrom organic bases including primary, secondary and tertiary amines,substituted amines including cyclic amines, such as caffeine, arginine,diethylamine, N-ethyl piperidine, aistidine, glucamine, isopropylamine,lysine, morpholine, N-ethyl morpholine, piperazine, piperidine,triethylamine, trimethylamine.

Additional examples of such acid and base addition salts can be found inBerge et al., J. Pharm. Sci., 66, 1 (1977). All of these salts may beprepared by conventional means from the corresponding compound of theinvention by reacting, for example, the appropriate acid or base withthe compound of Formula I.

Also, the basic nitrogen-containing groups of compounds of Formula I canbe quaternized with such agents as lower alkyl halides including,without limitation, methyl, ethyl, propyl, and butyl chloride, bromidesand iodides; dialkyl sulfates including dimethyl, diethyl, dibutyl, anddiamyl sulfates, long chain halides such as decyl, lauryl, myristyl andstearyl chlorides, bromides and iodides, aralkyl halides like benzyl andphenethyl bromides, and others. Water or oil-soluble or dispersibleproducts may be obtained by quaternizing such basic nitrogen groups incompounds of Formula I.

In general, “derivative” as used herein, refers to simple modifications,readily apparent to those of ordinary skill in the art, on the parentcore structure of Formula I, which does not significantly affect(generally decrease) the activity of the compound in-vitro as well as invivo, in a subject. The term, “derivative” as used herein, iscontemplated to include pharmaceutically acceptable derivatives ofcompounds of Formula I.

In general, “leaving group” as used herein, refers to groups readilydisplaceable by a nucleophile, such as an amine, a thiol or an alcoholnucleophile. Such leaving groups are well known in the art. Examples ofsuch leaving groups include, but are not limited to,N-hydroxysuccinimide, N-hydroxybenzotriazole, halides, triflates,tosylates and the like. Exemplary leaving groups are indicated hereinwhere appropriate.

In general, “protecting group” as used herein, refers to groups wellknown in the art which are used to prevent selected reactive groups,such as carboxy, amino, hydroxy, mercapto and the like, from undergoingundesired reactions, such as nucleophilic, electrophilic, oxidation,reduction and the like. Protecting groups are indicated herein whereappropriate.

Examples of amino protecting groups include, but are not limited to,aralkyl, substituted aralkyl, cycloalkenylalkyl and substitutedcycloalkenyl alkyl, allyl, substituted allyl, acyl, alkoxycarbonyl,aralkoxycarbonyl, silyl and the like. Examples of aralkyl include, butare not limited to, benzyl, ortho-methylbenzyl, trityl and benzhydryl,which can be optionally substituted with halogen, alkyl, alkoxy,hydroxy, nitro, acylamino, acyl and the like, and salts, such asphosphonium and ammonium salts. Examples of aryl groups include phenyl,naphthyl, indanyl, anthracenyl, 9-(9-phenylfluorenyl), phenanthrenyl,durenyl and the like. Examples of cycloalkenylalkyl or substitutedcycloalkylenylalkyl radicals, for example those having 6-10 carbonatoms, include, but are not limited to, cyclohexenyl methyl and thelike. Suitable acyl, alkoxycarbonyl and aralkoxycarbonyl groups includebenzyloxycarbonyl, t-butoxycarbonyl, iso-butoxycarbonyl, benzoyl,substituted benzoyl, butyryl, acetyl, tri-fluoroacetyl, tri-chloroacetyl, phthaloyl and the like. A mixture of protecting groups can beused to protect the same amino group, such as a primary amino group canbe protected by both an aralkyl group and an aralkoxycarbonyl group.Amino protecting groups can also form a heterocyclic ring with thenitrogen to which they are attached, for example,1,2-bis(methylene)benzene, phthalimidyl, succinimidyl, maleimidyl andthe like and where these heterocyclic groups can further includeadjoining aryl and cycloalkyl rings. In addition, the heterocyclicgroups can be mono-, di- or tri-substituted, such as nitrophthalimidyl.Amino groups may also be protected against undesired reactions, such asoxidation, through the formation of an addition salt, such ashydrochloride, toluenesulfonic acid, trifluoroacetic acid and the like.Many of the amino protecting groups, including aralkyl groups forexample, are also suitable for protecting carboxy, hydroxy and mercaptogroups. Alkyl groups are also suitable groups for protecting hydroxy andmercapto groups, such as tert-butyl.

Silyl protecting groups are groups containing silicon atoms which areoptionally substituted by one or more alkyl, aryl and aralkyl groups.Suitable silyl protecting groups include, but are not limited to,trimethylsilyl, triethylsilyl, tri-isopropylsilyl,tert-butyldimethylsilyl, dimethylphenylsilyl,1,2-bis(dimethylsilyl)benzene, 1,2-bis(dimethylsilyl)ethane anddiphenylmethylsilyl. Silylation of an amino groups provide mono- ordi-silylamino groups. Silylation of aminoalcohol compounds can lead to aN,N,O-tri-silyl derivative. Removal of the silyl function from a silylether function is readily accomplished by treatment with, for example, ametal hydroxide or ammonium fluoride reagent, either as a discretereaction step or in situ during a reaction with the alcohol group.Suitable silylating agents are, for example, trimethylsilyl chloride,tert-butyl-dimethylsilyl chloride, phenyldimethylsilyl chloride,diphenylmethyl silyl chloride or their combination products withimidazole or DMF. Methods for silylation of amines and removal of silylprotecting groups are well known to those skilled in the art. Methods ofpreparation of these amine derivatives from corresponding amino acids,amino acid amides or amino acid esters are also well known to thoseskilled in the art of organic chemistry including amino acid/amino acidester or aminoalcohol chemistry.

Protecting groups are removed under conditions which will not affect theremaining portion of the molecule. These methods are well known in theart and include acid hydrolysis, hydrogenolysis and the like. One methodinvolves removal of a protecting group, such as removal of abenzyloxycarbonyl group by hydrogenolysis utilizing palladium on carbonin a suitable solvent system such as an alcohol, acetic acid, and thelike or mixtures thereof. A t-butoxycarbonyl protecting group can beremoved utilizing an inorganic or organic acid, such as HCl ortrifluoroacetic acid, in a suitable solvent system, such as dioxane ormethylene chloride. The resulting amino salt can readily be neutralizedto yield the free amine. Carboxy protecting group, such as methyl,ethyl, benzyl, tert-butyl, 4-methoxyphenylmethyl and the like, can beremoved under hydrolysis and hydrogenolysis conditions well known tothose skilled in the art.

It should be noted that compounds of the invention may contain groupsthat may exist in tautomeric forms, such as cyclic and acyclic amidineand guanidine groups, heteroatom substituted heteroaryl groups (Y′=O, S,NR), and the like, which are illustrated in the following examples:

and though one form is named, described, displayed and/or claimedherein, all the tautomeric forms are intended to be inherently includedin such name, description, display and/or claim.

A. Derivatives

In addition to LCAT modifications described above, it is contemplatedthat other “derivatives” of LCAT may be substituted for an LCAT proteindescribed above. Such derivatives may improve the solubility,absorption, biological half life, and the like of the compounds. Themoieties may alternatively eliminate or attenuate any undesirableside-effect of the compounds and the like.

Such derivative LCATs include compounds in which:

1. The compound or some portion thereof is cyclic. For example, thepeptide portion may be modified to contain two or more cysteine residues(e.g., in the linker), which could cyclize by disulfide bond formation.

2. The compound is cross-linked or is rendered capable of cross-linkingbetween molecules. For example, the peptide portion may be modified tocontain one cysteine residue and thereby be able to form anintermolecular disulfide bond with a like molecule. The compound mayalso be cross-linked through its C-terminus.

3. One or more peptidyl [—C(O)NR—] linkages (bonds) is replaced by anon-peptidyl linkage. Exemplary non-peptidyl linkages are —CH₂-carbamate[—CH₂—OC(O)NR—], phosphonate, —CH₂-sulfonamide [—CH₂—S(O)₂NR—], urea[—NHC(O)NH—], —CH₂-secondary amine, and alkylated peptide [—C(O)NR₆—wherein R₆ is lower alkyl].

4. The N-terminus is derivatized. Typically, the N-terminus may beacylated or modified to a substituted amine. Exemplary N-terminalderivative groups include —NRR1 (other than —NH2), —NRC(O)R1,—NRC(O)OR1, —NRS(O)2R1, —NHC(O)NHR1, succinimide, orbenzyloxycarbonyl-NH— (CBZ—NH—), wherein R and R1 are each independentlyhydrogen or lower alkyl with the proviso that R and R1 are not bothhydrogen and wherein the phenyl ring may be substituted with 1 to 3substituents selected from the group consisting of C1-C4 alkyl, C1-C4alkoxy, chloro, and bromo; to a succinimide group; to abenzyloxycarbonyl-NH— (CBZ—NH—) group; and peptides wherein the free Cterminus is derivatized to —C(O)R2 where R2 is selected from the groupconsisting of lower alkoxy and —NR3R4 where R3 and R4 are independentlyselected from the group consisting of hydrogen and lower alkyl. By“lower” is meant a group having from 1 to 6 carbon atoms.

5. The free C-terminus is derivatized. Typically, the C-terminus isesterified or amidated. For example, one may use methods described inthe art to add (NH—CH2-CH2-NH2)₂ to compounds of this invention at theC-terminus. Likewise, one may use methods described in the art to add—NH2 to compounds of this invention at the C-terminus. ExemplaryC-terminal derivative groups include, for example, —C(O)R2 wherein R2 islower alkoxy or —NR3R4 wherein R3 and R4 are independently hydrogen orC1-C8 alkyl (preferably C1-C4 alkyl).

6. A disulfide bond is replaced with another, preferably more stable,cross-linking moiety (e.g., an alkylene). See, e.g., Bhatnagar et al.(1996), J. Med. Chem. 39: 3814-9; Alberts et al. (1993) Thirteenth Am.Pep. Symp., 357-9.

7. One or more individual amino acid residues is modified. Variousderivatizing agents are known to react specifically with selected sidechains or terminal residues, as described in detail below.

Additionally, modifications of individual amino acids may be introducedinto the LCAT amino acid sequence by reacting targeted amino acidresidues of the protein with an organic derivatizing agent that iscapable of reacting with selected side chains or terminal residues. Thefollowing are exemplary.

Lysinyl and amino terminal residues may be reacted with succinic orother carboxylic acid anhydrides. Derivatization with these agents hasthe effect of reversing the charge of the lysinyl residues. Othersuitable reagents for derivatizing alpha-amino-containing residuesinclude imidoesters such as methyl picolinimidate; pyridoxal phosphate;pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid;O-methylisourea; 2,4 pentanedione; and transaminase-catalyzed reactionwith glyoxylate.

Arginyl residues may be modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pKa of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineguanidino group.

The specific modification of tyrosyl residues per se has been studiedextensively, with particular interest in introducing spectral labelsinto tyrosyl residues by reaction with aromatic diazonium compounds ortetranitromethane. Most commonly, N-acetylmidizole and tetranitromethanemay be used to form O-acetyl tyrosyl species and 3-nitro derivatives,respectively.

Carboxyl side groups (aspartyl or glutamyl) may be selectively modifiedby reaction with carbodiimides (R′—N═C═N—R′) such as1-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide or1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore,aspartyl and glutamyl residues may be converted to asparaginyl andglutaminyl residues by reaction with ammonium ions.

Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutamyl and aspartyl residues. Alternatively, theseresidues may be deamidated under mildly acidic conditions. Either formof these residues falls within the scope of this invention.

Cysteinyl residues at a position other than residue 31 can be replacedby amino acid residues or other moieties either to eliminate disulfidebonding or, conversely, to stabilize cross-linking. See, e.g., Bhatnagaret al. (1996), J. Med. Chem. 39: 3814-9.

Derivatization with bifunctional agents is useful for cross-linking thepeptides or their functional derivatives to a water-insoluble supportmatrix or to other macromolecular carriers. Commonly used cross-linkingagents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane,glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with4-azidosalicylic acid, homobifunctional imidoesters, includingdisuccinimidyl esters such as 3,3′-dithiobis (succinimidylpropionate),and bifunctional maleimides such as bis-N-maleimido-1,8-octane.Derivatizing agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatableintermediates that are capable of forming crosslinks in the presence oflight. Alternatively, reactive water-insoluble matrices such as cyanogenbromide-activated carbohydrates and the reactive substrates described inU.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537;and 4,330,440 may be employed for protein immobilization.

Other possible modifications include hydroxylation of proline andlysine, phosphorylation of hydroxyl groups of seryl or threonylresidues, oxidation of the sulfur atom in cysteine, methylation of thealpha-amino groups of lysine, arginine, and histidine side chains(Creighton, T. E., Proteins: Structure and Molecule Properties, W. H.Freeman & Co., San Francisco, pp. 79-86 (1983)), acetylation of theN-terminal amine, and, in some instances, amidation of the C-terminalcarboxyl groups.

Such derivatized moieties preferably improve one or more characteristicsincluding thrombopoietic activity, solubility, absorption, biologicalhalf life, and the like of the inventive compounds. Alternatively,derivatized moieties result in compounds that have the same, oressentially the same, characteristics and/or properties of the compoundthat is not derivatized. The moieties may alternatively eliminate orattenuate any undesirable side effect of the compounds and the like.

Carbohydrate (oligosaccharide) groups may conveniently be attached tosites that are known to be glycosylation sites in proteins. Generally,O-linked oligosaccharides are attached to serine (Ser) or threonine(Thr) residues while N-linked oligosaccharides are attached toasparagine (Asn) residues when they are part of the sequenceAsn-X-Ser/Thr, where X can be any amino acid except proline. X ispreferably one of the 19 naturally occurring amino acids other thanproline. The structures of N-linked and O-linked oligosaccharides andthe sugar residues found in each type are different. One type of sugarthat is commonly found on both is N-acetylneuraminic acid (referred toas sialic acid). Sialic acid is usually the terminal residue of bothN-linked and O-linked oligosaccharides and, by virtue of its negativecharge, may confer acidic properties to the glycosylated compound. Suchsite(s) may be incorporated in the linker of the compounds of thisinvention and are preferably glycosylated by a cell during recombinantproduction of the polypeptide compounds (e.g., in mammalian cells suchas CHO, BHK, COS). However, such sites may further be glycosylated bysynthetic or semi-synthetic procedures known in the art.

Compounds of the present invention may be changed at the DNA level, aswell. The DNA sequence of any portion of the compound may be changed tocodons more compatible with the chosen host cell. For E. coli, which isthe host cell in one aspect, optimized codons are known in the art.Codons may be substituted to eliminate restriction sites or to includesilent restriction sites, which may aid in processing of the DNA in theselected host cell. The vehicle, linker and peptide DNA sequences may bemodified to include any of the foregoing sequence changes.

Isotope- and toxin-conjugated derivatives. Another set of usefulderivatives are the above-described molecules conjugated to toxins,tracers, or radioisotopes. Such conjugation is especially useful formolecules comprising peptide sequences that bind to tumor cells orpathogens. Such molecules may be used as therapeutic agents or as an aidto surgery (e.g., radioimmunoguided surgery or RIGS) or as diagnosticagents (e.g., radioimmunodiagnostics or RID).

As therapeutic agents, these conjugated derivatives possess a number ofadvantages. They facilitate use of toxins and radioisotopes that wouldbe toxic if administered without the specific binding provided by thepeptide sequence. They also can reduce the side-effects that attend theuse of radiation and chemotherapy by facilitating lower effective dosesof the conjugation partner.

Useful conjugation partners include:

-   -   radioisotopes, such as ⁹⁰Yttrium, ¹³¹Iodine, ²²⁵Actinium, and        ²¹³Bismuth;    -   ricin A toxin, microbially derived toxins such as Pseudomonas        endotoxin (e.g., PE38, PE40), and the like;    -   partner molecules in capture systems (see below);    -   biotin, streptavidin (useful as either partner molecules in        capture systems or as tracers, especially for diagnostic use);        and    -   cytotoxic agents (e.g., doxorubicin).

One useful adaptation of these conjugated derivatives is use in acapture system. In such a system, the molecule of the present inventionwould comprise a benign capture molecule. This capture molecule would beable to specifically bind to a separate effector molecule comprising,for example, a toxin or radioisotope. Both the vehicle-conjugatedmolecule and the effector molecule would be administered to the patient.In such a system, the effector molecule would have a short half-lifeexcept when bound to the vehicle-conjugated capture molecule, thusminimizing any toxic side-effects. The vehicle-conjugated molecule wouldhave a relatively long half-life but would be benign and non-toxic. Thespecific binding portions of both molecules can be part of a knownspecific binding pair (e.g., biotin, streptavidin) or can result frompeptide generation methods such as those described herein.

Such conjugated derivatives may be prepared by methods known in the art.In the case of protein effector molecules (e.g., Pseudomonas endotoxin),such molecules can be expressed as fusion proteins from correlative DNAconstructs. Radioisotope conjugated derivatives may be prepared, forexample, as described for the BEXA antibody (Coulter). Derivativescomprising cytotoxic agents or microbial toxins may be prepared, forexample, as described for the BR96 antibody (Bristol-Myers Squibb).Molecules employed in capture systems may be prepared, for example, asdescribed by the patents, patent applications, and publications fromNeoRx. Molecules employed for RIGS and RID may be prepared, for example,by the patents, patent applications, and publications from NeoProbe.

The compounds of the invention may also be covalently or noncovalentlyassociated with a carrier molecule, such as a linear polymer (e.g.,polyethylene glycol, polylysine, dextran, etc.), a branched-chainpolymer (see, for example, U.S. Pat. No. 4,289,872 to Denkenwalter etal., issued Sep. 15, 1981; U.S. Pat. No. 5,229,490 to Tam, issued Jul.20, 1993; WO 93/21259 by Frechet et al., published 28 Oct. 1993); alipid; a cholesterol group (such as a steroid); or a carbohydrate oroligosaccharide. Other possible carriers include one or more watersoluble polymer attachments such as polyoxyethylene glycol, orpolypropylene glycol as described U.S. Pat. Nos. 4,640,835, 4,496,689,4,301,144, 4,670,417, 4,791,192 and 4,179,337. Still other usefulpolymers known in the art include monomethoxy-polyethylene glycol,dextran, cellulose, or other carbohydrate based polymers, poly-(N-vinylpyrrolidone)-polyethylene glycol, propylene glycol homopolymers, apolypropylene oxide/ethylene oxide co-polymer, polyoxyethylated polyols(e.g., glycerol) and polyvinyl alcohol, as well as mixtures of thesepolymers.

In one aspect, the carrier is polyethylene glycol (PEG). The PEG groupmay be of any convenient molecular weight and may be straight chain orbranched. The average molecular weight of the PEG will range from about2 kDa to about 100 kDa, or from about 5 kDa to about 50 kDa, or fromabout 5 kDa to about 10 kDa.

The PEG groups will generally be attached to the compounds of theinvention via acylation, reductive alkylation, Michael addition, thiolalkylation or other chemoselective conjugation/ligation methods througha reactive group on the PEG moiety (e.g., an aldehyde, amino, ester,thiol, α-haloacetyl, maleimido or hydrazino group) to a reactive groupon the target compound (e.g., an aldehyde, amino, ester, thiol,α-haloacetyl, maleimido or hydrazino group).

Prodrugs of the compounds of this invention are also contemplated bythis invention. A “prodrug” is a compound, which when administered tothe body of a subject (such as a mammal), breaks down in the subject'smetabolic pathway to provide an active compound of Formula I. Morespecifically, a prodrug is an active or inactive “masked” compound thatis modified chemically through in vivo physiological action, such ashydrolysis, metabolism and the like, into a compound of this inventionfollowing administration of the prodrug to a subject or patient. Thesuitability and techniques involved in making and using prodrugs arewell known by those skilled in the art. For a general discussion ofprodrugs involving esters see Svensson and Tunek Drug Metabolism Reviews165 (1988) and Bundgaard Design of Prodrugs, Elsevier (1985).

One common form of a prodrug is a masked carboxylic acid group. Examplesof a masked carboxylate anion include a variety of esters, such as alkyl(for example, methyl, ethyl), cycloalkyl (for example, cyclohexyl),aralkyl (for example, benzyl, p-methoxybenzyl), andalkylcarbonyloxyalkyl (for example, pivaloyloxymethyl). Amines have beenmasked as arylcarbonyloxymethyl substituted derivatives which arecleaved by esterases in vivo releasing the free drug and formaldehyde(Bundgaard J. Med. Chem. 2503 (1989)). Also, drugs containing an acidicNH group, such as imidazole, imide, indole and the like, have beenmasked with N-acyloxymethyl groups (Bundgaard Design of Prodrugs,Elsevier (1985)). Hydroxy groups have been masked as esters and ethers.EP 039,051 (Sloan and Little, Apr. 11, 1981) discloses Mannich-basehydroxamic acid prodrugs, their preparation and use.

In general, “stereoisomer” as used herein refers to a compound havingone or more asymmetric centers. Chiral centers in a compound generallycause that compound to exist in many different conformations orstereoisomers. The term “stereoisomers” includes enantiomers,diastereomers, atropisomers and geometric isomers. Stereoisomersgenerally possess different chemical properties and/or biologicalactivity, as appreciated by those skilled in the art. For example, onestereoisomer may be more active and/or may exhibit beneficial effects incomparison to other stereoisomer(s) or when separated from the otherstereoisomer(s). However, it is well within the skill of the ordinaryartisan to separate, and/or to selectively prepare said stereoisomers.Accordingly, “stereoisomers” of the present invention necessarilyinclude mixtures of stereoisomers, including racemic mixtures,individual stereoisomers, and optically active forms.

In general, “solvate” when used with reference to a compound refers to acompound, which is associated with one or more molecules of a solvent,such as an organic solvent, inorganic solvent, aqueous solvent ormixtures thereof. The compounds of Formula I may also be solvated,especially hydrated. Hydration may occur during manufacturing of thecompounds or compositions comprising the compounds, or the hydration mayoccur over time due to the hygroscopic nature of the compounds.Compounds of the invention may exist as organic solvates as well,including DMF, ether, and alcohol solvates among others. Theidentification and preparation of any particular solvate is within theskill of the ordinary artisan of synthetic organic or medicinalchemistry.

B. Vehicles

1. Immunoglobulin Constant Region Vehicles

In one aspect, an LCAT protein of the invention includes at least onevehicle attached to the protein through the N-terminus, C-terminus or aside chain of one of the amino acid residues. In one embodiment, an Fcdomain is a vehicle. Thus, an Fc domain may be fused to the N or Ctermini of the peptides or at both the N and C termini. Multiplevehicles, as exemplified herein, may also be used; e.g., Fc's at eachterminus or an Fc at a terminus and a PEG group at the other terminus ora side chain.

In various embodiments, the Fc component is either a native Fc or an Fcvariant. By way of example and without limitation, the Fc component isan Fc region of the human immunoglobulin IgG1 heavy chain or abiologically active fragment, derivative, or dimer thereof, see Ellison,J. W. et al., Nucleic Acids Res. 10:4071-4079 (1982). It is understood,however, that an Fc region for use in the invention may be derived froman IgG, IgA, IgM, IgE or IgD from any species. Native Fc domains aremade up of monomeric polypeptides that may be linked into dimeric ormultimeric forms by covalent (i.e., disulfide bonds) and/or non-covalentassociation. The number of intermolecular disulfide bonds betweenmonomeric subunits of native Fc molecules ranges from 1 to 4 dependingon class (e.g., IgG, IgA, IgE) or subclass (e.g., IgG1, IgG2, IgG3,IgA1, IgGA2). One example of a native Fc is a disulfide-bonded dimerresulting from papain digestion of an IgG (see Ellison et al. (1982),Nucleic Acids Res. 10: 4071-9).

In various aspects, Fc sequence contemplated include those known in theart such as, for example, Fc IgG1 (GenBank Accession No. P01857), FcIgG2 (GenBank Accession No. P01859), Fc IgG3 (GenBank Accession No.P01860), Fc IgG4 (GenBank Accession No. P01861), Fc IgA1 (GenBankAccession No. P01876), Fc IgA2 (GenBank Accession No. P01877), Fc IgD(GenBank Accession No. P01880), Fc IgM (GenBank Accession No. P01871),and Fc IgE (GenBank Accession No. P01854).

Variants, analogs or derivatives of the Fc portion may be constructedby, for example, making various substitutions of residues or sequences.In one aspect, an Fc variant is incorporated which comprises a moleculeor sequence that is humanized from a non-human native Fc. Alternately,an Fc variant comprises a molecule or sequence that lacks one or morenative Fc sites or residues that affect or are involved in (1) disulfidebond formation, (2) incompatibility with a selected host cell (3)N-terminal heterogeneity upon expression in a selected host cell, (4)glycosylation, (5) interaction with complement, (6) binding to an Fcreceptor other than a salvage receptor, or (7) antibody-dependentcellular cytotoxicity (ADCC), each of which is described in detail inU.S. Patent Application No. 20040087778, the disclosure of which isincorporated by reference in its entirety.

Variant (or analog) polypeptides include insertion variants, wherein oneor more amino acid residues supplement an Fc amino acid sequence.Insertions may be located at either or both termini of the protein, ormay be positioned within internal regions of the Fc amino acid sequence.Insertion variants, with additional residues at either or both termini,can include for example, fusion proteins and proteins including aminoacid tags or labels. For example, the Fc molecule may optionally containan N-terminal Met, especially when the molecule is expressedrecombinantly in a bacterial cell such as E. coli.

In Fc deletion variants, one or more amino acid residues in an Fcpolypeptide are removed. Deletions can be effected at one or bothtermini of the Fc polypeptide, or with removal of one or more residueswithin the Fc amino acid sequence. Deletion variants, therefore, includeall fragments of an Fc polypeptide sequence.

In Fc substitution variants, one or more amino acid residues of an Fcpolypeptide are removed and replaced with alternative residues. In oneaspect, the substitutions are conservative in nature and conservativesubstitutions of this type are well known in the art. Alternatively, theinvention embraces substitutions that are also non-conservative.

For example, cysteine residues can be deleted or replaced with otheramino acids to prevent formation of some or all disulfide crosslinks ofthe Fc sequences. Each cysteine residue can be removed and/orsubstituted with other amino acids, such as Ala or Ser. As anotherexample, modifications may also be made to introduce amino acidsubstitutions to (1) ablate the Fc receptor binding site; (2) ablate thecomplement (C1q) binding site; and/or to (3) ablate the antibodydependent cell-mediated cytotoxicity (ADCC) site. Such sites are knownin the art, and any known substitutions are within the scope of Fc asused herein. For example, see Molecular Immunology, Vol. 29, No. 5,633-639 (1992) with regard to ADCC sites in IgG1.

Likewise, one or more tyrosine residues can be replaced by phenylalanineresidues. In addition, other variant amino acid insertions, deletionsand/or substitutions are also contemplated and are within the scope ofthe present invention. Conservative amino acid substitutions willgenerally be preferred. Furthermore, alterations may be in the form ofaltered amino acids, such as peptidomimetics or D-amino acids.

As noted above, both native Fcs and Fc variants are suitable Fc domainsfor use within the scope of this invention. A native Fc may beextensively modified to form an Fc variant provided binding to thesalvage receptor is maintained; see, for example WO 97/34631 and WO96/32478. In such Fc variants, one may remove one or more sites of anative Fc that provide structural features or functional activity notrequired by the fusion molecules of this invention. One may remove thesesites by, for example, substituting or deleting residues, insertingresidues into the site, or truncating portions containing the site. Theinserted or substituted residues may also be altered amino acids, suchas peptidomimetics or D-amino acids. Fc variants may be desirable for anumber of reasons, several of which are described below. Exemplary Fcvariants include molecules and sequences in which:

1. Sites involved in disulfide bond formation are removed. Such removalmay avoid reaction with other cysteine-containing proteins present inthe host cell used to produce the molecules of the invention. For thispurpose, the cysteine-containing segment at the N-terminus may betruncated or cysteine residues may be deleted or substituted with otheramino acids (e.g., alanyl, seryl). In particular, one may truncate theN-terminal 20-amino acid segment of SEQ ID NO: 3 or delete or substitutethe cysteine residues at positions 7 and 10 of SEQ ID NO: 3. Even whencysteine residues are removed, the single chain Fc domains can stillform a dimeric Fc domain that is held together non-covalently.

2. A native Fc is modified to make it more compatible with a selectedhost cell. For example, one may remove the PA sequence near theN-terminus of a typical native Fc, which may be recognized by adigestive enzyme in E. coli such as proline iminopeptidase. One may alsoadd an N-terminal methionine residue, especially when the molecule isexpressed recombinantly in a bacterial cell such as E. coli. The Fcdomain of SEQ ID NO: 3 is one such Fc variant.

3. A portion of the N-terminus of a native Fc is removed to preventN-terminal heterogeneity when expressed in a selected host cell. Forthis purpose, one may delete any of the first 20 amino acid residues atthe N-terminus, particularly those at positions 1, 2, 3, 4 and 5.

4. One or more glycosylation sites are removed. Residues that aretypically glycosylated (e.g., asparagine) may confer cytolytic response.Such residues may be deleted or substituted with unglycosylated residues(e.g., alanine).

5. Sites involved in interaction with complement, such as the Cl qbinding site, are removed. For example, one may delete or substitute theEKK sequence of human IgG 1.

Complement recruitment may not be advantageous for the molecules of thisinvention and so may be avoided with such an Fc variant.

6. Sites are removed that affect binding to Fc receptors other than asalvage receptor. A native Fc may have sites for interaction withcertain white blood cells that are not required for the fusion moleculesof the present invention and so may be removed.

7. The ADCC site is removed. ADCC sites are known in the art; see, forexample, Molec. Immunol. 29 (5): 633-9 (1992) with regard to ADCC sitesin IgG1. These sites, as well, are not required for the fusion moleculesof the present invention and so may be removed.

8. When the native Fc is derived from a non-human antibody, the nativeFc may be humanized. Typically, to humanize a native Fc, one willsubstitute selected residues in the non-human native Fc with residuesthat are normally found in human native Fc. Techniques for antibodyhumanization are well known in the art.

It should be noted that Fc monomers will spontaneously dimerize when theappropriate cysteine residues are present, unless particular conditionsare present that prevent dimerization through disulfide bond formation.Even if the cysteine residues that normally form disulfide bonds in theFc dimer are removed or replaced by other residues, the monomeric chainswill generally form a dimer through non-covalent interactions. The term“Fc” herein is used to mean any of these forms: the native monomer, thenative dimer (disulfide bond linked), modified dimers (disulfide and/ornon-covalently linked), and modified monomers (i.e., derivatives).

Fc sequences may also be derivatized, i.e., bearing modifications otherthan insertion, deletion, or substitution of amino acid residues. In oneaspect, the modifications are covalent in nature, and include forexample, chemical bonding with polymers, lipids, other organic, andinorganic moieties. However, non-covalent modifications are alsocontemplated. Derivatives of the invention may be prepared to increasecirculating half-life, or may be designed to improve targeting capacityfor the polypeptide to desired cells, tissues, or organs.

It is also possible to use the salvage receptor binding domain of theintact Fc molecule as the Fc part of a compound of the invention, suchas described in WO 96/32478, entitled “Altered Polypeptides withIncreased Half-Life.” Additional members of the class of moleculesdesignated as Fc herein are those that are described in WO 97/34631,entitled “Immunoglobulin-Like Domains with Increased Half-Lives.” Bothof the published PCT applications cited in this paragraph are herebyincorporated by reference.

As discussed herein, the Fc fusions may be at the N or C terminus of aTMP of the invention, or at both the N and C termini of the TMP. It hasbeen previously been shown that peptides in which an Fc moiety isligated to the N terminus of the TMP group is more bioactive than theother possibilities. When the Fc is fused at the N-terminus of the TMPor linker, such fusion will generally occur at the C-terminus of the Fcchain, and vice versa.

2. Water-Soluble Polymer Vehicles

As noted above, polymer vehicles are also contemplated. Various meansfor attaching chemical moieties useful as vehicles are currentlyavailable, see, e.g., Patent Cooperation Treaty (“PCT”) InternationalPublication No. WO 96/11953, entitled “N-Terminally Chemically ModifiedProtein Compositions and Methods,” herein incorporated by reference inits entirety. This PCT publication discloses, among other things, theselective attachment of water soluble polymers to the N-terminus ofproteins.

Thus, the invention contemplates compounds comprising a water-solublepolymer (WSP). Suitable, clinically acceptable, WSP include withoutlimitation, PEG, polyethylene glycol propionaldehyde, copolymers ofethylene glycol/propylene glycol, monomethoxy-polyethylene glycol,carboxymethylcellulose, polyacetals, polyvinyl alcohol (PVA), polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleicanhydride copolymer, poly (.beta.-amino acids) (either homopolymers orrandom copolymers), poly(n-vinyl pyrrolidone)polyethylene glycol,propropylene glycol homopolymers (PPG) and other polyakylene oxides,polypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols(POG) (e.g., glycerol) and other polyoxyethylated polyols,polyoxyethylated sorbitol, or polyoxyethylated glucose, colonic acids orother carbohydrate polymers, Ficoll or dextran and mixtures thereof. Infact, any of the forms of PEG that have been used to derivatize otherproteins, such as and without limitation mono-(C1-C10) alkoxy- oraryloxy-polyethylene glycol, are provided. Polyethylene glycolpropionaldehyde may have advantages in manufacturing due to itsstability in water.

The PEG group may be of any convenient molecular weight and may belinear or branched. The average molecular weight of PEG contemplated foruse in the invention ranges from about 2 kDa to about 100 kDa, fromabout 5 kDa to about 50 kDa, from about 5 kDa to about 10 kDa. Inanother aspect, the PEG moiety has a molecular weight from about 6 kDato about 25 kDa. PEG groups generally are attached to peptides orproteins via acylation or reductive alkylation through a reactive groupon the PEG moiety (e.g., an aldehyde, amino, thiol, or ester group) to areactive group on the target peptide or protein (e.g., an aldehyde,amino, or ester group). Using methods described herein, a mixture ofpolymer/peptide conjugate molecules can be prepared, and the advantageprovided herein is the ability to select the proportion ofpolymer/peptide conjugate to include in the mixture. Thus, if desired, amixture of peptides with various numbers of polymer moieties attached(i.e., zero, one or two) can be prepared with a predetermined proportionof polymer/protein conjugate.

A useful strategy for the PEGylation of synthetic peptides consists ofcombining, through forming a conjugate linkage in solution, a peptideand a WSP (PEG) moiety, each bearing a special functionality that ismutually reactive toward the other. The peptides can be easily preparedwith conventional solid phase synthesis. The peptides are “preactivated”with an appropriate functional group at a specific site. The precursorsare purified and fully characterized prior to reacting with the PEGmoiety. Ligation of the peptide with PEG usually takes place in aqueousphase and can be easily monitored by reverse phase analytical HPLC. ThePEGylated peptides can be easily purified by preparative HPLC andcharacterized by analytical HPLC, amino acid analysis and laserdesorption mass spectrometry.

Polysaccharide polymers are another type of WSP which may be used forprotein modification. Dextrans are polysaccharide polymers comprised ofindividual subunits of glucose predominantly linked by α1-6 linkages.The dextran itself is available in many molecular weight ranges, and isreadily available in molecular weights from about 1 kD to about 70 kD.Dextran is a suitable water soluble polymer for use in the presentinvention as a vehicle by itself or in combination with another vehicle(e.g., Fc). See, for example, WO 96/11953 and WO 96/05309. The use ofdextran conjugated to therapeutic or diagnostic immunoglobulins has beenreported; see, for example, European Patent Publication No. 0 315 456,which is hereby incorporated by reference. Dextran of about 1 kD toabout 20 kD is preferred when dextran is used as a vehicle in accordancewith the present invention.

The WSP moiety of the molecule may be branched or unbranched. Fortherapeutic use of the end-product preparation, the polymer ispharmaceutically acceptable. In general, a desired polymer is selectedbased on such considerations as whether the polymer conjugate will beused therapeutically, and if so, the desired dosage, circulation time,resistance to proteolysis, and other considerations. In various aspects,the average molecular weight of each WSP is between about 2 kDa andabout 100 kDa, between about 5 kDa and about 50 kDa, between about 12kDa and about 40 kDa and between about 20 kDa and about 35 kDa. In yetanother aspect the molecular weight of each polymer is between about 6kDa and about 25 kDa. The term “about” as used herein and throughout,indicates that in preparations of a water soluble polymer, somemolecules will weigh more, some less, than the stated molecular weight.Generally, the higher the molecular weight or the more branches, thehigher the polymer/protein ratio. Other sizes may be used, depending onthe desired therapeutic profile including for example, the duration ofsustained release; the effects, if any, on biological activity; the easein handling; the degree or lack of antigenicity and other known effectsof a water soluble polymer on a therapeutic protein.

The WSP should be attached to the protein with consideration given toeffects on functional or antigenic domains of the peptide or protein. Ingeneral, chemical derivatization may be performed under any suitablecondition used to react a protein with an activated polymer molecule.Activating groups which can be used to link the water soluble polymer toone or more proteins include without limitation sulfone, maleimide,sulfhydryl, thiol, triflate, tresylate, azidirine, oxirane and5-pyridyl. If attached to the peptide by reductive alkylation, thepolymer selected should have a single reactive aldehyde so that thedegree of polymerization is controlled.

3. Alternative Vehicles

Alternative vehicles include a protein, polypeptide, peptide, antibody,antibody fragment, or small molecule (e.g., a peptidomimetic compound)capable of binding to a salvage receptor. For example, one could use asa vehicle a polypeptide as described in U.S. Pat. No. 5,739,277, issuedApr. 14, 1998 to Presta et al. Peptides could also be selected by phagedisplay for binding to the FcRn salvage receptor. Such salvagereceptor-binding compounds are also included within the meaning of“vehicle” and are within the scope of this invention. Such vehiclesshould be selected for increased half-life (e.g., by avoiding sequencesrecognized by proteases) and decreased immunogenicity (e.g., by favoringnon-immunogenic sequences, as discovered in antibody humanization).

Compounds that are useful in the methods of the instant invention are,for example, compounds of Formula I

or a stereomer, a tautomer, a solvate, a pharmaceutically acceptablesalt, or a prodrug thereof, wherein X, Y and Z are independentlyselected from the group consisting of —N═, —S—, —CH═ and

provided that at least two of X, Y and Z are not —S—, and provided thatno more than one of X, Y and Z is —CH═; L is —S—, —S(O)—, or —S(O)₂—;

R¹ is selected from the group consisting of CN, COOR⁵, SO₂R⁶ andhalogen;

R² is selected from the group consisting of H, optionally substitutedC₁-C₁₂ alkyl, optionally substituted C₁-C₁₂ alkenyl, optionallysubstituted C₁-C₈ alkynyl, optionally substituted aryl, and optionallysubstituted heteroaryl, and SR³, wherein the substituents are selectedfrom the group consisting of C₁-C₄ alkyl, NH₂, halo and CN; and whereinR³ is selected from the group consisting of H, optionally substitutedC₁-C₁₂ alkyl, optionally substituted C₁-C₈ alkenyl, optionallysubstituted C₁-C₈ alkynyl, optionally substituted aryl and optionallysubstituted heteroaryl, wherein the substituents are selected from thegroup consisting of NH₂, halo and CN;

R⁴ is H or C₁-C₈ alkyl;

R⁵ and R⁶ are each independently C₁-C₄ alkyl.

In one aspect, X and Y are each —N═.

In another aspect, Z is —S—.

In a further aspect, L is —S—.

In one aspect, R¹ is CN.

In another aspect, R² is SR³. R³ can be C₁-C₄ alkyl, for example,methyl.

In one aspect, the invention contemplates the use of the followingcompounds:

-   3-(5-(Methylthio)-1,3,4-thiadiazol-2-ylthio)pyrazine-2-carbonitrile;-   3-(5-(Ethylthio)-1,3,4-thiadiazol-2-ylthio)pyrazine-2-carbonitrile;-   3-(5-(Allylthio)-1,3,4-thiadiazol-2-ylthio)pyrazine-2-carbonitrile;-   3-(5-(Propylthio)-1,3,4-thiadiazol-2-ylthio)pyrazine-2-carbonitrile;-   3-(5-(Butylthio)-1,3,4-thiadiazol-2-ylthio)pyrazine-2-carbonitrile;-   3-(5-(Isobutylthio)-1,3,4-thiadiazol-2-ylthio)pyrazine-2-carbonitrile;-   3-(5-(Pentylthio)-1,3,4-thiadiazol-2-ylthio)pyrazine-2-carbonitrile;-   3-(5-(Dodecylthio)-1,3,4-thiadiazol-2-ylthio)pyrazine-2-carbonitrile;-   3-(5-(Benzylthio)-1,3,4-thiadiazol-2-ylthio)pyrazine-2-carbonitrile;-   3-(5-Mercapto-1,3,4-thiadiazol-2-ylthio)pyrazine-2-carbonitrile;-   3-(5-(Isopropylthio)-4-methyl-4H-1,2,4-triazol-3-ylthio)pyrazine-2-carbonitrile;-   3-(5-(Methylthio)-1,2,4-thiadiazol-3-ylthio)pyrazine-2-carbonitrile;-   3-(5-Methyl-1,3,4-thiadiazol-2-ylthio)pyrazine-2-carbonitrile;-   3-(5-Butyl-1,3,4-thiadiazol-2-ylthio)pyrazine-2-carbonitrile;-   3-(4-Methyl-4H-1,2,4-triazol-3-ylthio)pyrazine-2-carbonitrile;-   3-(1-Methyl-1H-imidazol-2-ylthio)pyrazine-2-carbonitrile;-   2-Chloro-3-(5-(methylthio)-1,3,4-thiadiazol-2-ylthio)pyrazine-   or a pharmaceutically acceptable salt thereof.

In one aspect, the invention provides the modified LCAT polypeptide,wherein the amino acid residue 31 is replaced by a cysteine residue,wherein the cysteine residue is modified by replacing the thiol hydrogenwith 3-pyrazinyl-2-carbonitrile.

In another aspect, the invention contemplates the use of apharmaceutical composition comprising the modified LCAT polypeptide,wherein the amino acid residue 31 is replaced by a cysteine residue,wherein the cysteine residue is modified by replacing the thiol hydrogenwith 3-pyrazinyl-2-carbonitrile, and a pharmaceutically acceptablecarrier.

Preparation of Compounds

The compounds of the present invention can be prepared using standardsynthetic methods. For exemplary purposes, Scheme 1 illustrates methodsfor the preparation of compounds of structural formula (III). One ofskill in the art will understand that similar methods can be used forthe synthesis of compounds in the other structural classes.

As shown in Scheme 1, compounds of the present invention can be preparedbeginning with the commercially available 2-chloropyrazinecarbonitrile(I). Treatment of I with a thiol, such as II in the presence of basesuch as NaH, K₂CO₃ or CsCO₃ in a suitable solvent such as THF, DMF orDMSO provides the adduct (III). Oxidation of the thio group in III withfor example H₂O₂, oxone, or MnO₂ will give the sulfone or sulfoxidederivative. Alternatively, other oxidizing agents may be employed asdescribed in March, J; Advanced Organic Chemistry, 5th ed., John Wiley &Sons, New York, pp. 1541 (2001).

Other compounds of the present invention can be prepared beginning with2,3-dichloropyrazine IV as shown in Scheme 2. Treatment of IV with athiol, such as II in the presence of base such as NaH, K₂CO₃ or CsCO₃ ina suitable solvent such as THF, DMF or DMSO provides the adduct V.Compound V can also be converted to compounds of formula III bytreatment with, for example, potassium cyanide or zinc cyanide in thepresence of a palladium catalyst in a suitable solvent such as THF orDMF (see e.g. Y. Akita et al, Synthesis, 974, (1981)).

Preparation of the compounds of the invention is described in moredetail in Examples below.

III. Pharmaceutical Compositions Comprising Modified LCAT and Methods ofAdministration

While it may be possible to administer compounds of the invention alone,in the methods described, the compound administered is generally presentas an active ingredient in a desired dosage unit formulation, such aspharmaceutically acceptable composition containing conventionalpharmaceutically acceptable carriers. Thus, in another aspect of theinvention, there is provided a pharmaceutical composition comprising acompound of this invention in combination with a pharmaceuticallyacceptable carrier. Acceptable pharmaceutical carriers generally includediluents, excipients, adjuvants and the like as described herein.

A pharmaceutical composition of the invention may comprise an effectiveamount of a compound of the invention or an effective dosage amount of acompound of the invention. An effective dosage amount of a compound ofthe invention includes an amount less than, equal to, or greater than aneffective amount of the compound. For example, a pharmaceuticalcomposition in which two or more unit dosages, such as in tablets,capsules and the like, are required to administer an effective amount ofthe compound, or alternatively, a multi-dose pharmaceutical composition,such as powders, liquids and the like, in which an effective amount ofthe compound may be administered by administering a portion of thecomposition. “Unit dosage” is defined as a discrete amount of atherapeutic composition dispersed in a suitable carrier. Those ofordinary skill in the art will readily optimize effective dosages andadministration regimens as determined by good medical practice and theclinical condition of the individual patient.

The pharmaceutical compositions may generally be prepared by mixing oneor more compounds of Formula I including stereoisomers or tautomers,solvates, pharmaceutically acceptable salts, derivatives or prodrugsthereof, with pharmaceutically acceptable carriers, excipients, binders,adjuvants, diluents and the like, to form a desired administrableformulation to treat or ameliorate a variety of disorders related toatherosclerosis or cardiovascular diseases.

The pharmaceutical compositions may generally be prepared by mixing oneor more LCAT compounds with one or more pharmaceutically acceptablecarriers, excipients, binders, adjuvants, diluents, preservatives,solubilizers, emulsifiers and the like, to form a desired administrableformulation to treat or ameliorate a variety of diseases. Suchcompositions include diluents of various buffer content (e.g., Tris-HCl,acetate, phosphate), pH and ionic strength; additives such as detergentsand solubilizing agents (e.g., Tween 80, Polysorbate 80), anti-oxidants(e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g.,Thimersol, benzyl alcohol) and bulking substances (e.g., lactose,mannitol); incorporation of the material into particulate preparationsof polymeric compounds such as polylactic acid, polyglycolic acid, etc.or into liposomes. Hyaluronic acid may also be used, and this may havethe effect of promoting sustained duration in the circulation. Suchcompositions may influence the physical state, stability, rate of invivo release, and rate of in vivo clearance of the present proteins andderivatives. See, e.g., Remington's Pharmaceutical Sciences, 18th Ed.(1990, Mack Publishing Co., Easton, Pa. 18042) pages 1435-1712 which areherein incorporated by reference. The compositions may be prepared inliquid form, or may be in dried powder, such as lyophilized form.Implantable sustained release formulations are also contemplated, as aretransdermal formulations.

The pharmaceutical compositions may be subjected to conventionalpharmaceutical operations such as sterilization and/or may containconventional adjuvants, such as preservatives, stabilizers, wettingagents, emulsifiers, buffers etc. The pharmaceutically active compoundsof this invention can be processed in accordance with conventionalmethods of pharmacy to produce medicinal agents for administration topatients, including humans and other mammals.

Pharmaceutical compositions can be manufactured by methods well known inthe art such as conventional granulating, mixing, dissolving,encapsulating, lyophilizing, emulsifying or levigating processes, amongothers. The compositions can be in the form of, for example, granules,powders, tablets, capsules, syrup, suppositories, injections, emulsions,elixirs, suspensions or solutions. The instant compositions can beformulated for various routes of administration, for example, by oraladministration, by transmucosal administration (including pulmonary andnasal administration), parenteral administration (including subcutaneousadministration), transdermal (topical) administration or by rectaladministration, as well as intrathecal, intravenous, intramuscular,intraperitoneal, intranasal, intraocular or intraventricular injection.The compound or compounds of the instant invention can also beadministered in a local rather than a systemic fashion, such asinjection as a sustained release formulation.

Besides those representative dosage forms described herein,pharmaceutically acceptable excipients and carriers are generally knownto those skilled in the art and are thus included in the instantinvention. Such excipients and carriers are described, for example, in“Remingtons Pharmaceutical Sciences” Mack Pub. Co., New Jersey (2000);and “Pharmaceutics The Science of Dosage Form Design, 2^(nd) Ed.(Aulton, ed.) Churchill Livingstone (2002). The following dosage formsare given by way of example and should not be construed as limiting theinvention.

A. Oral Administration

For oral, buccal, and sublingual administration, powders, suspensions,granules, tablets, pills, capsules, gelcaps, troches or lozenges,cachets, pellets and caplets are acceptable as solid dosage (and unitdosage) forms and are described generally in Chapter 89 of Remington'sPharmaceutical Sciences (1990), 18th Ed., Mack Publishing Co. Easton Pa.18042, which is herein incorporated by reference. Solid dosage formsalso include liposomal or proteinoid encapsulation (for example,proteinoid microspheres reported in U.S. Pat. No. 4,925,673). Liposomalencapsulation may be used and the liposomes may be derivatized withvarious polymers (e.g., U.S. Pat. No. 5,013,556). A description ofpossible solid dosage forms for the therapeutic is given in Chapter 10of Marshall, K., Modern Pharmaceutics (1979), edited by G. S. Banker andC. T. Rhodes, herein incorporated by reference. In general, theformulation includes the LCAT compound, and inert ingredients whichallow for protection against the stomach environment, and release of thebiologically active material in the intestine.

If necessary, the compounds are chemically modified to enhancebioefficacy of oral delivery. Generally, the chemical modificationcontemplated is the attachment of at least one moiety to the compoundmolecule itself, where said moiety permits (a) inhibition ofproteolysis; and (b) uptake into the blood stream from the stomach orintestine. Also desired is the increase in overall stability of thecompound and increase in circulation time in the body. Moieties usefulas covalently attached vehicles in this invention may also be used forthis purpose. Examples of such moieties include: PEG, copolymers ofethylene glycol and propylene glycol, carboxymethyl cellulose, dextran,polyvinyl alcohol, polyvinyl pyrrolidone and polyproline as well asother moieties described herein. See also, for example, Abuchowski andDavis, Soluble Polymer-Enzyme Adducts, Enzymes as Drugs (1981),Hocenberg and Roberts, eds., Wiley-Interscience, New York, N.Y., pp367-83; Newmark, et al. (1982), J. Appl. Biochem. 4:185-9. Otherpolymers that could be used are poly-1,3-dioxolane andpoly-1,3,6-tioxocane. In one aspect, PEG moieties are provided forpharmaceutical usage, as indicated above.

For oral delivery dosage forms, it is also possible to use a salt of amodified aliphatic amino acid, such as sodiumN-(8-[2-hydroxybenzoyl]amino) caprylate (SNAC), as a carrier to enhanceabsorption of the therapeutic compounds of this invention. The clinicalefficacy of a heparin formulation using SNAC has been demonstrated in aPhase II trial conducted by Emisphere Technologies. See U.S. Pat. No.5,792,451, “Oral drug delivery composition and methods”.

The compounds of this invention can be included in the formulation asfine multiparticulates in the form of granules or pellets of particlesize about 1 mm. The formulation of the material for capsuleadministration could also be as a powder, lightly compressed plugs oreven as tablets. The therapeutic could be prepared by compression.

Oral pharmaceutical compositions contemplated can be prepared, forexample, by mixing one or more compounds of the instant invention, orstereoisomers, solvates, prodrugs, pharmaceutically acceptable salts ortautomers thereof, with at least one additive or excipient such as astarch or other additive and tableted, encapsulated or made into otherdesirable forms for conventional administration. Suitable additives orexcipients are sucrose, lactose, cellulose sugar, mannitol, maltitol,dextran, sorbitol, starch, agar, alginates, chitins, chitosans, pectins,tragacanth gum, gum arabic, gelatins, collagens, casein, albumin,synthetic or semi-synthetic polymers or glycerides, methyl cellulose,hydroxypropylmethyl-cellulose, and/or polyvinylpyrrolidone. Optionally,oral dosage forms can contain other ingredients to aid inadministration, such as an inactive diluent, or lubricants such asmagnesium stearate, or preservatives such as paraben or sorbic acid, oranti-oxidants such as ascorbic acid, tocopherol or cysteine, adisintegrating agent, binders, thickeners, buffers, sweeteners,flavoring agents or perfuming agents. Additionally, dyestuffs orpigments may be added for identification. Tablets and pills may befurther treated with suitable coating materials known in the art.

Liquid dosage forms for oral administration may be in the form ofpharmaceutically acceptable emulsions, syrups, elixirs, suspensions,slurries and solutions, which may contain an inactive diluent, such aswater. Pharmaceutical formulations may be prepared as liquid suspensionsor solutions using a sterile liquid, such as, but not limited to, anoil, water, an alcohol, and combinations of these. Pharmaceuticallysuitable surfactants, suspending agents, emulsifying agents, and thelike may be added for oral or parenteral administration. Morespecifically, various aspects of oral pharmaceutical compositionsinclude one or more of the following additives.

Colorants and flavoring agents may all be included. For example, theprotein (or derivative) may be formulated (such as by liposome ormicrosphere encapsulation) and then further contained within an edibleproduct, such as a refrigerated beverage containing colorants andflavoring agents.

One may dilute or increase the volume of the compound of the inventionwith an inert material. These diluents could include carbohydrates,especially mannitol, -lactose, anhydrous lactose, cellulose, sucrose,modified dextrans and starch. Certain inorganic salts may also be usedas fillers including calcium triphosphate, magnesium carbonate andsodium chloride. Some commercially available diluents are Fast-Flo,Emdex, STA-Rx 1500, Emcompress and Avicell.

Disintegrants may be included in the formulation of the therapeutic intoa solid dosage form. Materials used as disintegrants include but are notlimited to starch including the commercial disintegrant based on starch,Explotab. Sodium starch glycolate, Amberlite, sodiumcarboxymethylcellulose, ultramylopectin, sodium alginate, gelatin,orange peel, acid carboxymethyl cellulose, natural sponge and bentonitemay all be used. Another form of the disintegrants are the insolublecationic exchange resins. Powdered gums may be used as disintegrants andas binders and these can include powdered gums such as agar, Karaya ortragacanth. Alginic acid and its sodium salt are also useful asdisintegrants.

Binders may be used to hold the therapeutic agent together to form ahard tablet and include materials from natural products such as acacia,tragacanth, starch and gelatin. Others include methyl cellulose (MC),ethyl cellulose (EC) and carboxymethyl cellulose (CMC). Polyvinylpyrrolidone (PVP) and hydroxypropylmethyl cellulose (HPMC) could both beused in alcoholic solutions to granulate the therapeutic.

An antifrictional agent may be included in the formulation of thetherapeutic to prevent sticking during the formulation process.Lubricants may be used as a layer between the therapeutic and the diewall, and these can include but are not limited to; stearic acidincluding its magnesium and calcium salts, polytetrafluoroethylene(PTFE), liquid paraffin, vegetable oils and waxes. Soluble lubricantsmay also be used such as sodium lauryl sulfate, magnesium laurylsulfate, polyethylene glycol of various molecular weights, Carbowax 4000and 6000.

Glidants that might improve the flow properties of the drug duringformulation and to aid rearrangement during compression might be added.The glidants may include starch, talc, pyrogenic silica and hydratedsilicoaluminate.

To aid dissolution of the compound of this invention into the aqueousenvironment a surfactant might be added as a wetting agent. Surfactantsmay include anionic detergents such as sodium lauryl sulfate, dioctylsodium sulfosuccinate and dioctyl sodium sulfonate. Cationic detergentsmight be used and could include benzalkonium chloride or benzethoniumchloride. The list of potential nonionic detergents that could beincluded in the formulation as surfactants are lauromacrogol 400,polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fattyacid ester, methyl cellulose and carboxymethyl cellulose. Thesesurfactants could be present in the formulation of the protein orderivative either alone or as a mixture in different ratios.

Additives may also be included in the formulation to enhance uptake ofthe compound. Additives potentially having this property are forinstance the fatty acids oleic acid, linoleic acid and linolenic acid.

Controlled release formulation may be desirable. The compound of thisinvention could be incorporated into an inert matrix which permitsrelease by either diffusion or leaching mechanisms e.g., gums. Slowlydegenerating matrices may also be incorporated into the formulation,e.g., alginates, polysaccharides. Another form of a controlled releaseof the compounds of this invention is by a method based on the Orostherapeutic system (Alza Corp.), i.e., the drug is enclosed in asemipermeable membrane which allows water to enter and push drug outthrough a single small opening due to osmotic effects. Some entericcoatings also have a delayed release effect.

Other coatings may be used for the formulation. These include a varietyof sugars which could be applied in a coating pan. The therapeutic agentcould also be given in a film coated tablet and the materials used inthis instance are divided into 2 groups. The first are the nonentericmaterials and include methyl cellulose, ethyl cellulose, hydroxyethylcellulose, methylhydroxy-ethyl cellulose, hydroxypropyl cellulose,hydroxypropyl-methyl cellulose, sodium carboxy-methyl cellulose,providone and the polyethylene glycols. The second group consists of theenteric materials that are commonly esters of phthalic acid.

A mix of materials might be used to provide the optimum film coating.Film coating may be carried out in a pan coater or in a fluidized bed orby compression coating.

B. Pulmonary Delivery Forms

Also contemplated herein is pulmonary delivery of the present protein(or derivatives thereof). The protein (or derivative) is delivered tothe lungs of a mammal while inhaling and traverses across the lungepithelial lining to the blood stream. (Other reports of this includeAdjei et al., Pharma. Res. (1990) 7: 565-9; Adjei et al. (1990),Internatl. J. Pharmaceutics 63: 135-44 (leuprolide acetate); Braquet etal. (1989), J. Cardiovasc. Pharmacol. 13 (suppl. 5): s.143-146(endothelin-1); Hubbard et al. (1989), Annals Int. Med. 3: 206-12(α1-antitrypsin); Smith et al. (1989), J. Clin. Invest. 84: 1145-6(α1-proteinase); Oswein et al. (March 1990), “Aerosolization ofProteins”, Proc. Symp. Resp. Drug Delivery II, Keystone, Colo.(recombinant human growth hormone); Debs et al. (1988), J. Immunol. 140:3482-8 (interferon-γ and tumor necrosis factor α) and Platz et al., U.S.Pat. No. 5,284,656 (granulocyte colony stimulating factor).

Contemplated for use in the practice of this invention are a wide rangeof mechanical devices designed for pulmonary delivery of therapeuticproducts, including but not limited to nebulizers, metered doseinhalers, and powder inhalers, all of which are familiar to thoseskilled in the art. Some specific examples of commercially availabledevices suitable for the practice of this invention are the Ultraventnebulizer, manufactured by Mallinckrodt, Inc., St. Louis, Mo.; the AcornII nebulizer, manufactured by Marquest Medical Products, Englewood,Colo.; the Ventolin metered dose inhaler, manufactured by Glaxo Inc.,Research Triangle Park, North Carolina; and the Spinhaler powderinhaler, manufactured by Fisons Corp., Bedford, Mass.

All such devices require the use of formulations suitable for thedispensing of the inventive compound. Typically, each formulation isspecific to the type of device employed and may involve the use of anappropriate propellant material, in addition to diluents, adjuvantsand/or carriers useful in therapy.

The inventive compound should most advantageously be prepared inparticulate form with an average particle size of less than 10 m (ormicrons), most preferably 0.5 to 5 m, for most effective delivery to thedistal lung.

Pharmaceutically acceptable carriers for pulmonary delivery includecarbohydrates such as trehalose, mannitol, xylitol, sucrose, lactose,and sorbitol. Other ingredients for use in formulations may includeDPPC, DOPE, DSPC and DOPC. Natural or synthetic surfactants may be used.PEG may be used (even apart from its use in derivatizing the protein oranalog). Dextrans, such as cyclodextran, may be used. Bile salts andother related enhancers may be used. Cellulose and cellulose derivativesmay be used. Amino acids may be used, such as use in a bufferformulation.

Also, the use of liposomes, microcapsules or microspheres, inclusioncomplexes, or other types of carriers is contemplated.

Formulations suitable for use with a nebulizer, either jet orultrasonic, will typically comprise the inventive compound dissolved inwater at a concentration of about 0.1 to 25 mg of biologically activeprotein per mL of solution. The formulation may also include a bufferand a simple sugar (e.g., for protein stabilization and regulation ofosmotic pressure). The nebulizer formulation may also contain asurfactant, to reduce or prevent surface induced aggregation of theprotein caused by atomization of the solution in forming the aerosol.

Formulations for use with a metered-dose inhaler device will generallycomprise a finely divided powder containing the inventive compoundsuspended in a propellant with the aid of a surfactant. The propellantmay be any conventional material employed for this purpose, such as achlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or ahydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane,dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, orcombinations thereof. Suitable surfactants include sorbitan trioleateand soya lecithin. Oleic acid may also be useful as a surfactant.

Formulations for dispensing from a powder inhaler device will comprise afinely divided dry powder containing the inventive compound and may alsoinclude a bulking agent, such as lactose, sorbitol, sucrose, mannitol,trehalose, or xylitol in amounts which facilitate dispersal of thepowder from the device, e.g., 50 to 90% by weight of the formulation.

C. Nasal Administration

Nasal delivery of the inventive compound is also contemplated. Nasaldelivery allows the passage of the protein to the blood stream directlyafter administering the therapeutic product to the nose, without thenecessity for deposition of the product in the lung. Formulations fornasal delivery include those with dextran or cyclodextran. Delivery viatransport across other mucous membranes is also contemplated.

For nasal administration, the pharmaceutical formulations may be a sprayor aerosol containing an appropriate solvent and optionally othercompounds such as, but not limited to, stabilizers, antimicrobialagents, antioxidants, pH modifiers, surfactants, bioavailabilitymodifiers and combinations of these. A propellant for an aerosolformulation may include compressed air, nitrogen, carbon dioxide, or ahydrocarbon based low boiling solvent. The compound or compounds of theinstant invention are conveniently delivered in the form of an aerosolspray presentation from a nebulizer or the like.

D. Parenteral Administration

Injectable dosage forms for parenteral administration generally includeaqueous suspensions or oil suspensions, which may be prepared using asuitable dispersant or wetting agent and a suspending agent. Injectableforms may be in solution phase or a powder suitable for reconstitutionas a solution. Both are prepared with a solvent or diluent. Acceptablesolvents or vehicles include sterilized water, Ringer's solution, or anisotonic aqueous saline solution. Alternatively, sterile oils may beemployed as solvents or suspending agents. Typically, the oil or fattyacid is non-volatile, including natural or synthetic oils, fatty acids,mono-, di- or tri-glycerides. For injection, the formulations mayoptionally contain stabilizers, pH modifiers, surfactants,bioavailability modifiers and combinations of these. The compounds maybe formulated for parenteral administration by injection such as bybolus injection or continuous infusion. A unit dosage form for injectionmay be in ampoules or in multi-dose containers.

E. Rectal Administration

For rectal administration, the pharmaceutical formulations may be in theform of a suppository, an ointment, an enema, a tablet or a cream forrelease of compound in the intestines, sigmoid flexure and/or rectum.Rectal suppositories are prepared by mixing one or more compounds of theinstant invention, or pharmaceutically acceptable salts or tautomers ofthe compound, with acceptable vehicles, for example, cocoa butter orpolyethylene glycol, which is solid phase at room temperature but liquidphase at those temperatures suitable to release a drug inside the body,such as in the rectum. Various other agents and additives may be used inthe preparation of suppositories as is well known to those of skill inthe art.

F. Forms

The formulations of the invention may be designed to be short-acting,fast-releasing, long-acting, and sustained-releasing as described below.Thus, the pharmaceutical formulations may also be formulated forcontrolled release or for slow release. The instant compositions mayalso comprise, for example, micelles or liposomes, or some otherencapsulated form, or may be administered in an extended release form toprovide a prolonged storage and/or delivery effect. Therefore, thepharmaceutical formulations may be compressed into pellets or cylindersand implanted intramuscularly or subcutaneously as depot injections oras implants such as stents. Such implants may employ known inertmaterials such as silicones and biodegradable polymers.

G. Dosages

Specific dosages may be adjusted depending on conditions of disease, theage, body weight, general health conditions, sex, and diet of thesubject, dose intervals, administration routes, excretion rate, andcombinations of drugs. Any of the above dosage forms containingeffective amounts are well within the bounds of routine experimentationand therefore, well within the scope of the instant invention.

A therapeutically effective dose may vary depending upon the route ofadministration and dosage form. Typically, the compound or compounds ofthe instant invention are selected to provide a formulation thatexhibits a high therapeutic index. The therapeutic index is the doseratio between toxic and therapeutic effects which can be expressed asthe ratio between LD₅₀ and ED₅₀. The LD₅₀ is the dose lethal to 50% ofthe population and the ED₅₀ is the dose therapeutically effective in 50%of the population. The LD₅₀ and ED₅₀ are determined by standardpharmaceutical procedures in animal cell cultures or experimentalanimals.

The dosage regimen for treating LCAT-mediated diseases and otherdiseases listed above with the compounds of this invention and/orcompositions of this invention is based on a variety of factors,including the type of disease, the age, weight, sex, medical conditionof the patient, the severity of the condition, the route ofadministration, and the particular compound employed. Thus, the dosageregimen may vary widely, but can be determined routinely using standardmethods. Dosage levels of the order from about 0.01 mg to 30 mg perkilogram of body weight per day, for example from about 0.1 mg to 10mg/kg, or from about 0.25 mg to 1 mg/kg are useful for all methods ofuse disclosed herein. Generally, the daily regimen should be in therange of 0.1-1000 micrograms of the compound per kilogram of bodyweight, preferably 0.1-150 micrograms per kilogram.

For oral administration, the pharmaceutical composition may be in theform of, for example, a capsule, a tablet, a suspension, or liquid. Thepharmaceutical composition can be made in the form of a dosage unitcontaining a given amount of the active ingredient. For example, thesemay contain an amount of active ingredient from about 1 to 2000 mg, forexample from about 1 to 500 mg, or from about 5 to 150 mg, or from 10 to100 mg. A suitable daily dose for a human or other mammal may varywidely depending on the condition of the patient and other factors, but,once again, can be determined using routine methods.

The active ingredient may also be administered by injection as acomposition with suitable carriers including saline, dextrose, or water.The daily parenteral dosage regimen will be from about 0.1 to about 30mg/kg of total body weight, such as from about 0.1 to about 10 mg/kg, orfrom about 0.25 mg to 1 mg/kg.

Formulations suitable for topical administration include liquid orsemi-liquid preparations suitable for penetration through the skin(e.g., liniments, lotions, ointments, creams, or pastes) and dropssuitable for administration to the eye, ear, or nose.

A suitable topical dose of active ingredient of a compound of theinvention is 0.1 mg to 150 mg administered one to four, for example oneor two times daily. For topical administration, the active ingredientmay comprise from 0.001% to 10% w/w, e.g., from 1% to 2% by weight ofthe formulation, although it may comprise as much as 10% w/w, buttypically not more than 5% w/w. In one aspect, the concentration is from0.1% to 1% of the formulation.

H. Administration Regimens

Administration of the compositions can be systemic or local, and maycomprise a single site injection of a therapeutically-effective amountof the modified LCAT polypeptide composition. Any route known to thoseof skill in the art for the administration of a therapeutic compositionof the invention is contemplated including, for example, intravenous,intramuscular, subcutaneous or a catheter for long-term administration.Alternatively, it is contemplated that the therapeutic composition maybe delivered to the patient at multiple sites. The multipleadministrations may be rendered simultaneously or may be administeredover a period of several hours. In certain cases, it may be beneficialto provide a continuous flow of the therapeutic composition. Additionaltherapy may be administered on a period basis, for example, daily,weekly, or monthly. In certain embodiments, the modified LCATpolypeptide is provided locally to the site of reperfusion.

IV. Methods of Treatment

A. Atherosclerosis, Cardiovascular Disease or an Associated Disease

In one aspect, the method of treatment of the invention is therapeutic,and compounds and compositions of the invention are administered to asubject already suffering from atherosclerosis, cardiovascular diseaseor an associated disease. In another aspect, methods of treatment areprophylactic and compounds and compositions are administered to thosesubjects at risk for developing atherosclerosis. To determine whether asubject is at risk of, for example atherosclerosis, an atherogeniclipoprotein profile can be assessed. For example, a ratio of serumcholesterol to HDLs of 5:1 or above indicates a higher than average riskof developing atherosclerosis. Other factors include a serum cholesterollevel of 240 mg/dL or above, an HDL level 35 mg/dL or below, or an LDLlevel 190 mg/dL or above, a plasma LCAT protein level lower than normal(<5 ug/ml), and a decreased plasma cholesterol esterification rate (<60nmol/ml/hr).

The amount of the modified LCAT effective to decrease accumulation ofcholesterol depends on several factors, including the species, themanner of administration, the general health of the subject, the desiredresult (e.g., prophylaxis or therapeutic treatment) and the judgment ofthe prescribing physician. For example, the practitioner may decide whatrisk levels for heart disease indicate prophylactic treatment, and whattarget level of the modified LCAT is indicated for the treatment of aperson already suffering from atherosclerosis.

In humans, the normal cholesterol esterification rate ranges from about60 nmol/ml/hr to about 130 nmol/mL per hour. The effective treatment ofatherosclerosis in humans can involve administration of the compositionsof the invention to achieve a cholesterol esterification rate of about200 nmol/ml/hr.

The invention provides methods for the treatment, prevention, ormanagement of a cardiovascular disease. As used herein, the term“cardiovascular diseases” refers to diseases of the heart andcirculatory system. Cardiovascular diseases which the compositions ofthe present invention are useful for preventing or treating include butare not limited to arteriosclerosis; atherosclerosis; stroke; ischemia;endothelium dysfunctions, in particular those dysfunctions affectingblood vessel elasticity; peripheral vascular disease; coronary heartdisease; myocardial infarction, cerebral infarction and restenosis,thrombosis, high blood pressure and angina. Other diseases which thecompositions of the present invention are useful for preventing ortreating include LCAT deficiency syndrome, Alzheimer's disease, cornealopacity, metabolic syndrome, dyslipidemia, myocardial infarction,stroke, critical limb ischemia.

B. Inflammatory Conditions

Methods, compounds and compositions of the invention are useful insuppressing inflammatory cell activation. The term “inflammatory cellactivation,” as used herein, means the induction by a stimulus(including, but not limited to, cytokines, antigens or auto-antibodies)of a proliferative cellular response, the production of solublemediators (including but not limited to cytokines, oxygen radicals,enzymes, prostanoids, or vasoactive amines), or cell surface expressionof new or increased numbers of mediators (including, but not limited to,major histocompatability antigens or cell adhesion molecules) ininflammatory cells (including but not limited to monocytes, macrophages,T lymphocytes, B Lymphocytes, granulocytes, polymorphonuclearleukocytes, mast cells, basophils, eosinophils, dendritic cells, andendothelial cells). It will be appreciated by persons skilled in the artthat the activation of one or a combination of these phenotypes in thesecells can contribute to the initiation, perpetuation, or exacerbation ofan inflammatory condition.

Methods, compounds and compositions of the invention are useful intreating such diseases as arthritic diseases (such as rheumatoidarthritis), osteoarthritis, gouty arthritis, spondylitis,thyroid-associated ophthalmopathy, Behcet disease, sepsis, septic shock,endotoxic shock, gram negative sepsis, gram positive sepsis, toxic shocksyndrome, asthma, chronic bronchitis, allergic rhinitis, allergicconjunctivitis, vernal conjunctivitis, eosinophilic granuloma, adult(acute) respiratory distress syndrome (ARDS), chronic pulmonaryinflammatory disease (such as chronic obstructive pulmonary disease),silicosis, pulmonary sarcoidosis, reperfusion injury of the myocardium,brain or extremities, brain or spinal cord injury due to minor trauma,fibrosis including cystic fibrosis, keloid formation, scar tissueformation, atherosclerosis, autoimmune diseases, such as systemic lupuserythematosus (SLE) and transplant rejection disorders (e.g., graft vs.host (GvH) reaction and allograft rejection), chronicglomerulonephritis, inflammatory bowel diseases, such as Crohn's diseaseand ulcerative colitis, proliferative lymphocytic diseases, such asleukemias (e.g. chronic lymphocytic leukemia; CLL) (see Munoz et al., J.Exp. Med. 172:95-103 (1990); Mentz et al., Blood 88:2172-2182 (1996)),and inflammatory dermatoses, such as atopic dermatitis, psoriasis, orurticaria.

C. Thrombosis-Related Conditions

It is also contemplated that the compounds, compositions and methods ofthe present invention are used in the treatment of a variety ofdisorders in which there is a need to prevent or treat thrombosis andsubsequent decrease or loss of blood flow. The examples of thromoboticdisorders include but not limited to atherosclerosis, myocardialinfarction, stroke, and kidney ischemia, and thrombosis in any part ofthe mammalian body. The composition of the present invention will alsobe used in the prevention and treatment of microangiopathy in whichformation of microthrombi or von Willebrand factor (VWF) binding toplatelets causes excessive consumption of platelets and/or VWF leadingto subsequent bleeding diathesis. Examples of latter disorders includebut not limited to thrombotic thrombocytopenic purpura, type II andplatelet type von Willebrand disease (VWD). The compounds or combinationtherapeutic methods of the present invention inhibit VWF-dependentplatelet adhesion and aggregation. The compounds, compositions andmethods are also useful in prolonging bleed time in a mammal and assuch, are useful as anti-thrombotic agents both in therapeutic andprophylactic methods. As such, these compounds, compositions and methodsare useful as anticoagulant agents and/or anti-platelet agents. Further,the present invention provides compounds, compositions and methods forthe treatment of thrombosis and other disorders of the cardiovascularcirculatory system that require and increase in the flow or reducingblockage of the vessels.

Compounds, compositions and methods are also useful for the treatment ofany disorder that is presently treated using anticoagulant therapy. Suchdisorders include pulmonary embolism, unstable angina, myocardialinfarction, deep vein thrombosis, atrial fibrillation with embolization,acute and chronic coagulopathies (disseminated intravascularcoagulation), for prevention of clotting in arterial and cardiacsurgery, for prophylaxis and treatment of peripheral arterial embolism,The compounds, compositions and methods are also used to treatthrombotic thrombocytopic purpura, other types of microangiopathy thatare mediated by spontaneous interaction between VWF and platelets,platelet type or type IIb von Willebrand diseases in which there is anincreased binding of VWF to platelets (either caused by a defect in GPIbor in VWF). The compounds, compositions and methods described herein areuseful as anti-platelet agents in blood transfusions, extracorporealcirculation, dialysis procedures as well as blood sampling forlaboratory procedures. The compounds, compositions and methods are alsoused to maintain the patency of an indwelling venipucture device that isbeing used for intermittent injection or infusion therapy or bloodsampling. The compounds, compositions and methods are particularlyuseful in surgical procedures to prevent the formation of blood clots.Such indications are particularly desirable for patients undergoingabdominal surgery to reduce the risk of thromboemolic complications,patients undergoing knee or hip replacement therapy during and followingthe replacement procedure, as well as a general prophylactic to preventclot formation at a later stage. The compounds, compositions and methodsare further useful in the treatment of subjects that are under risk ofthromboembolic complications due to severely restricted mobility e.g.,during acute illness. Any such disorders may be readily treated by thecompositions described herein. The therapeutic methods include bothmedical therapeutic and/or prophylactic administration, as appropriate.

As used herein, the term “inhibits platelet aggregation” includes itsgenerally accepted meaning which includes prohibiting, slowing, orreducing the severity or degree of platelet aggregation. Such aninhibition may be measured as a function of time taken for a givensample to coagulate. In other embodiments, animal models of thrombosis.Methods of determining the efficacy of the agents include coagulationtesting, monitoring the time of bleeding, determining hemoglobin levelsof an animal and the like.

V. Combination Therapy

The invention further provides combination therapy, wherein thecompounds and/or compositions of the invention are administered with oneor more additional agent(s) In general, the therapeutic methods,compositions and compounds may also be employed in combination withother therapeutics in the treatment of various disease states, with theadditional agents being administered concurrently or sequentially with acomposition of the invention.

A. Cytokines

Exemplary cytokines or hematopoietic factors for such co-administrationinclude IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-11,colony stimulating factor-1 (CSF-1), M-CSF, SCF, GM-CSF, granulocytecolony stimulating factor (G-CSF), EPO, interferon-alpha (IFN-alpha),consensus interferon, IFN-beta, IFN-gamma, IFN-omega, IL-7, IL-8, IL-9,IL-10, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20,IL-21, IL-22, IL-23, IL-24, IL-31, IL-32 alpha, IL-33, thrombopoietin(TPO), angiopoietins, for example Ang-1, Ang-2, Ang-4, Ang-Y, the humanangiopoietin-like polypeptides ANGPTL1 through 7, vitronectin, vascularendothelial growth factor (VEGF), angiogenin, activin A, activin B,activin C, bone morphogenic protein-1, bone morphogenic protein-2, bonemorphogenic protein-3, bone morphogenic protein-4, bone morphogenicprotein-5, bone morphogenic protein-6, bone morphogenic protein-7, bonemorphogenic protein-8, bone morphogenic protein-9, bone morphogenicprotein-10, bone morphogenic protein-11, bone morphogenic protein-12,bone morphogenic protein-13, bone morphogenic protein-14, bonemorphogenic protein-15, bone morphogenic protein receptor IA, bonemorphogenic protein receptor IB, bone morphogenic protein receptor II,brain derived neurotrophic factor, cardiotrophin-1, ciliary neutrophicfactor, ciliary neutrophic factor receptor, cripto, cryptic,cytokine-induced neutrophil chemotactic factor 1, cytokine-inducedneutrophil, chemotactic factor 2α, cytokine-induced neutrophilchemotactic factor 2β,β endothelial cell growth factor, endothelin 1,epidermal growth factor, epigen, epiregulin, epithelial-derivedneutrophil attractant, fibroblast growth factor 4, fibroblast growthfactor 5, fibroblast growth factor 6, fibroblast growth factor 7,fibroblast growth factor 8, fibroblast growth factor 8b, fibroblastgrowth factor 8c, fibroblast growth factor 9, fibroblast growth factor10, fibroblast growth factor 11, fibroblast growth factor 12, fibroblastgrowth factor 13, fibroblast growth factor 16, fibroblast growth factor17, fibroblast growth factor 19, fibroblast growth factor 20, fibroblastgrowth factor 21, fibroblast growth factor acidic, fibroblast growthfactor basic, glial cell line-derived neutrophic factor receptor al,glial cell line-derived neutrophic factor receptor α2, growth relatedprotein, growth related protein a, growth related protein β, growthrelated protein γ, heparin binding epidermal growth factor, hepatocytegrowth factor, hepatocyte growth factor receptor, hepatoma-derivedgrowth factor, insulin-like growth factor I, insulin-like growth factorreceptor, insulin-like growth factor II, insulin-like growth factorbinding protein, keratinocyte growth factor, leukemia inhibitory factor,leukemia inhibitory factor receptor α, nerve growth factor nerve growthfactor receptor, neuropoietin, neurotrophin-3, neurotrophin-4,oncostatin M (OSM), placenta growth factor, placenta growth factor 2,platelet-derived endothelial cell growth factor, platelet derived growthfactor, platelet derived growth factor A chain, platelet derived growthfactor AA, platelet derived growth factor AB, platelet derived growthfactor B chain, platelet derived growth factor BB, platelet derivedgrowth factor receptor a, platelet derived growth factor receptor β,pre-B cell growth stimulating factor, stem cell factor (SCF), stem cellfactor receptor, TNF, including TNF0, TNF1, TNF2, transforming growthfactor α, transforming growth factor β, transforming growth factor β1,transforming growth factor β1.2, transforming growth factor β2,transforming growth factor β3, transforming growth factor β5, latenttransforming growth factor β1, transforming growth factor β bindingprotein I, transforming growth factor β binding protein II, transforminggrowth factor β binding protein III, thymic stromal lymphopoietin(TSLP), tumor necrosis factor receptor type I, tumor necrosis factorreceptor type II, urokinase-type plasminogen activator receptor,vascular endothelial growth factor, and chimeric proteins andbiologically or immunologically active fragments thereof.

B. Atherosclerosis Drugs

Additional active agents may act in complementary or synergistic wayswith the modified LCAT when used to treat, and prevent atherosclerosisor manage cholesterol, or related disorders such as cardiovasculardisease.

In one aspect, compounds of the invention can be used with statins.Statins are drugs that competitively inhibit 3-hydroxy-3-methylglutarylcoenzyme A reductase “HMG-CoA reductase,” which is the enzyme thatcatalyzes an early, rate-limiting step in cholesterol biosynthesis.Hebert et al., JAMA 1997, 278: 313-21. This combination, in addition toraising HDL levels and lowering LDL levels may also lowers triglycerideand reduce inflammation. It is believed that the combination can haveadditional therapeutic effects, for example, the combination may lowerblood pressure; protect against heart disease, for example, by reducingsmooth muscle proliferation, reduce heart attacks, reduce plateletaggregation, and to reduce strokes as well as peripheral arterialdisease (clogging of the arteries to the legs).

Examples of statins of the invention include, but are not limited to,mevastatin, pitavastatin, rosuvastatin, pentostatin (Nipent®), nystatin,lovastatin (Mevacor®), simvastatin (Zocor®), pravastatin (Pravachol®),fluvastatin (Lescol®), atorvastatin (Lipitor®), cerivastatin (Baycol®),or combinations thereof. Statins suitable for use in the compositionsand methods of the invention are also disclosed in U.S. Pat. Nos.4,681,893; 5,273,995; 5,356,896; 5,354,772; 5,686,104; 5,969,156; and6,126,971. As some statins may exist in an inactive form, such as alactone (e.g., simvastatin), the invention encompasses using the activeform (e.g., b-hydroxy acid form) of them. See Physicians Desk Reference,54.sup.th Ed. (2000) pp. 1917-1920.

Fibrates or fibric acid derivatives are regarded as broad-spectrumlipid-modulating agents in that although their main action is todecrease serum triglycerides they also tend to reduce LDL-cholesteroland to raise HDL-cholesterol. It is believed that the combined use ofcompounds of the invention and a fibrate may reduce the risk of coronaryheart disease events in those with low HDL-cholesterol or with raisedtriglycerides by speeding up the chemical breakdown (i.e., catabolism)of triglyceride-rich lipoproteins that circulate in the body.

Fibrates include, but are not limited to, bezafibrate, ciprofibrate,fenofibrate, gemfibrozil, clofibrate, or combinations thereof. Fibratessuitable for inclusion in the compositions or administration in themethods of the invention are disclosed in U.S. Pat. Nos. 4,895,762;6,074,670; and 6,277,405.

Biguanides for use in the compositions and methods of the inventioninclude, but are not limited to, metformin, phenformin, buformin, orcombinations thereof. Biguanides suitable for use in the compositions ormethods of the invention are also disclosed in U.S. Pat. No. 6,303,146.The combined use of compounds of the invention and a bigaunide mayimprove glycemic control by enhancing insulin sensitivity in the liverand in muscle. The combination may reduce or avoid cardiovascular riskfactors such as dyslipidemia, elevated plasminogen activator inhibitor 1levels, other fibrinolytic abnormalities, hyperinsulinemia, insulinresistance, and is an effective and safe therapeutic agent for thetreatment of type 2 diabetes.

In another aspect, compounds of the invention may be used in combinationwith glitazones, which may increase glucose uptake in muscle and reducedendogenous glucose production. Glitazones include5-((4-(2-(methyl-2-pyri-dinylamino)ethoxy)-phenyl)methyl)-2,4-thiazolidinedione, troglitazone,pioglitazone, ciglitazone, WAY-120,744, englitazone, AD 5075,darglitazone, rosiglitazone, combinations thereof, or a pharmaceuticallyacceptable salt, solvate, clathrate, polymorph, prodrug, orpharmacologically active metabolite thereof. Glitazones suitable for usein the compositions or methods of the invention are disclosed in U.S.Pat. Nos. 4,687,777; 5,002,953; 5,741,803; 5,965,584; 6,150,383;6,150,384; 6,166,042; 6,166,043; 6,172,090; 6,211,205; 6,271,243;6,288,095; 6,303,640; and 6,329,404.

Compositions comprising compounds of the invention and a sulfonylurea ora derivative thereof may increase insulin release from the pancreas andmay further insulin levels by reducing hepatic clearance of the hormone.Sulfonylurea-based drugs for use the compositions and methods of theinvention include, but are not limited to, glisoxepid, glyburide,acetohexamide, chlorpropamide, glibomuride, tolbutamide, tolazamide,glipizide, gliclazide, gliquidone, glyhexamide, phenbutamide,tolcyclamide, combinations thereof, or a pharmaceutically acceptablesalt, solvate, or clathrate.

Combination compositions may also include agents that inhibit CETP. Suchagents are, for example, Torcetrapib, and S-(2[([1-(2-ethylbutyl)cyclohexyl]carbonyl)amino]phenyl)-2-methylpropanethioate.

Additional active agents also include cardiovascular drugs.Cardiovascular drugs for use in combination with the compounds of theinvention to prevent or treat cardiovascular diseases include peripheralantiadrenergic drugs, centrally acting antihypertensive drugs (e.g.,methyldopa, methyldopa HCl), antihypertensive direct vasodilators (e.g.,diazoxide, hydralazine HCl), drugs affecting renin-angiotensin system,peripheral vasodilators, phentolamine, antianginal drugs, cardiacglycosides, inodilators (e.g., aminone, milrinone, enoximone,fenoximone, imazodan, sulmazole), antidysrhythmic drugs, calcium entryblockers, ranitine, bosentan, and rezulin.

Depending on the disorder for which treatment is sought, compounds andcompositions of the invention are used in combination therapy with othertherapeutics that achieve a specific biological effect.

1. Cholesterol Lowering Drugs

Various medications can lower blood cholesterol levels. They may beprescribed individually or in combination with other drugs. Some of thecommon types of cholesterol-lowering drugs include statins, resins andnicotinic acid (niacin), gemfibrozil and clofibrate. Thus, combinationtherapy is contemplated utilizing, for example, clofibrate (Atromid-S,which raises the HDL cholesterol levels and lowers triglyceride levels),gemfibrozil (Lopid, which raises HDL cholesterol levels), nicotinic acid(which works in the liver by affecting the production of blood fats andis used to lower triglycerides and LDL cholesterol, and raise HDL(“good”) cholesterol), resins (which are also called bile acid-bindingdrugs and work in the intestines by promoting increased disposal ofcholesterol), including cholestyramine (Questran, Prevalite,Lo-Cholest), colestipol (Colestid) and colesevelam (WelChol), andstatins including atorvastatin (Lipitor), fluvastatin (Lescol),lovastatin (Mevacor), pravastatin (Pravachol), rosuvastatin calcium(Crestor), and simvastatin (Zocor).

The drugs of first choice for elevated LDL cholesterol are the HMG CoAreductase inhibitors, e.g., atorvastatin, fluvastatin, lovastatin,pravastatin, rosuvastatin and simvastatin. Statin drugs are effectivefor lowering LDL cholesterol levels, have few immediate short-term sideeffects, are easy to administer, have high patient acceptance and havefew drug-drug interactions.

Another class of drugs for lowering LDL is the bile acidsequestrants—colesevelam, cholestyramine and colestipol—and nicotinicacid (niacin), which have been shown to reduce the risk for coronaryheart disease in controlled clinical trials. Both classes of drugsappear to be free of serious side effects. But both can have troublesomeside effects and require considerable patient education to achieveadherence. Nicotinic acid is preferred in patients with triglyceridelevels that exceed 250 mg/dL because bile acid sequestrants tend toraise triglyceride levels.

2. ACE Inhibitors

Angiotensin II causes blood vessels to contract and thereby narrows theblood vessels. The narrowing of the vessels increases the pressurewithin the vessels and can cause high blood pressure (hypertension).Angiotensin II is formed from angiotensin I in the blood by the enzyme,angiotensin converting enzyme (ACE). ACE inhibitors decrease theproduction of angiotensin II. As a result, the blood vessels enlarge ordilate, and the blood pressure is reduced. ACE inhibitors that availablein the United States include captopril (Capoten), benazepril (Lotensin),enalapril (Vasotec), lisinopril (Prinivil, Zestril) fosinopril(Monopril), ramipril (Altace), perindopril (Aceon), quinapril(Accupril), moexipril (Univasc), and trandolapril (Mavik).

C. Anti-Inflammatory Drugs

In prevention and treatment of inflammation, combination therapy iscontemplated with for example, acetylsalicylic acid (Aspirin, Ecotrin),choline magnesium salicylate (Trilisate), diclofenac (Voltaren,Cataflam, Voltaren-XR), diflunisal (Dolobid), etodolac (Lodine),fenoprofen (Nalfon), flurbiprofen (Ansaid), ibuprofen (Advil, Motrin,Medipren, Nuprin), indomethacin (Indocin, Indocin-SR), ketoprofen(Orudis, Oruvail), meclofenamate (Meclomen), nabumetone (Relafen),naproxen (Naprosyn, Naprelan, Anaprox, Aleve), oxaprozin (Daypro),phenylbutazone (Butazolidine), piroxicam (Feldene), salsalate (Disalcid,Salflex), tolmetin (Tolectin), valdecoxib (Bextra), and COX-2 selectivenon-steroidal anti-inflammatory drugs (NSAIDs) including Bextra,Celebrex, Naproxen, and Vioxx. Prescription-only NSAIDs includeibuprofen (Brufen), aceclofenac (Preservex), acemetacin (Emflex),azapropazone (Rheumox), celecoxib (Celebrex), dexketoprofen (Keral),diclofenac (Voltarol, Diclomax, Arthrotec), diflusinal (Dolobid),etodolac (Lodine), fenbufen (Lederfen), fenoprofen (Fenopron),flurbiprofen (Froben), indometacin, ketoprofen (Orudis, Oruvail),mefenamic acid, meloxicam (Mobic), nabumetone (Relifex), naproxen(Naprosyn, Synflex), phenylbutazone (Butacote), piroxicam (Feldene),sulindac (Clinoril), tenoxicam (Mobiflex) and tiaprofenic acid (Surgam),

D. Anti-Thrombosis Drugs

In methods for prevention and treatment of thrombosis-relatedconditions, combination therapy is contemplated with anti-thrombosisdrugs such as anticoagulant drugs, which inhibit the ability of blood toclot, or coagulate and include dalteparin (Fragmin), danaparoid(Orgaran), enoxaparin (Lovenox), heparin (various), tinzaparin(Innohep), warfarin (Coumadin), and lepirudin (Refludan), andantiplatelet drugs such as aspirin, ticlopidine (Ticlid), clopidogrel(Plavix), tirofiban (Aggrastat) and eptifibatide (Integrilin). Stillother methods include the use of bivalirudin (selective and reversiblethrombin inhibitor), argatroban (reversible inhibitor of thrombin), andlow molecular weight heparins (LMWHs), including enoxaparin (Lovenox),dalteparin (Fragmin), ardeparin (Normiflo) fondaparinux and idraparinux.Still other anti-thrombosis drugs contemplated for use in methods of theinvention include fragmin (dalteparin sodium injection) lovenox(enoxaparin sodium), Normiflo (ardeparin sodium), Orgaran (danaparoidsodium), indirect (Antithrombin-Dependent) FXa inhibitors such asfondaparinux (Arixtra®) and idraparinux, direct(Antithrombin-Independent) FXa inhibitors such as BAY 59-7939 [Bayer],DPC-423 [Bristol-Myers Squibb], DX-9065a [Daiichi], LY517717, razaxaban(DPC906), lepirudin (Refludan®), desirudin (Revasc®), bivalirudin(Hirulog®, Angiomax®), argatroban (Novastan®), melagatran, andximelagatran (Exanta®).

It should be understood that the disorder that may be treated by thecompositions of the present invention are limited only by the fact thatthe disorder needs a therapeutic intervention which inhibits plateletaggregation. The doses of the agent may be modified for each individualsubject. For particular guidance on the routes of administration, anduses those of skill in the art are referred to the Physician's DeskReference for generalized descriptions of formulations, routes ofadministration and patient monitoring used for agents such as Aggrastat™(see e.g., entry at pages 1933-1937, PDR, 57th Edn., 2003), Aggrenox™(see e.g., entry at pages 1023-1026, PDR, 57th Edn., 2003), Agrylin™(see e.g., entry at pages 3142-3143, PDR, 57th Edn., 2003), Flolan™ (seee.g., entry at pages 1516-1521, PDR, 57th Edn., 2003), Integrilin™ (seee.g., entry at pages 2138-2142, PDR, 57th Edn., 2003), Presantine™ (seee.g., entry at pages 1052-2053, PDR, 57th Edn., 2003), Plavix™ (seee.g., entry at pages 1098-1101, PDR, 57th Edn., 2003), Pletal™ (seee.g., entry at pages 2780-2782, PDR, 57th Edn., 2003), REoPro™ (seee.g., entry at pages 1866-1870, PDR, 57th Edn., 2003), Coumdin™ (seee.g., entry at pages 1074-1079, PDR, 57th Edn., 2003), Fragmin™ (seee.g., entry at pages 2750-2754, PDR, 57th Edn., 2003), Hep-Lock™ (seee.g., entry at pages 1284-1288, PDR, 57th Edn., 2003), Lovenox™ (seee.g., entry at pages 739-744, PDR, 57th Edn., 2003), Miradon™ (see e.g.,entry at pages 3051-3052, PDR, 57th Edn., 2003). These entries in thePDR are provided to show the level of skill in the art relating toformulating and using compositions as anticoagulants and anti-plateletagents.

It is understood that the application of the teachings of the presentinvention to a specific problem or situation will be within thecapabilities of one having ordinary skill in the art in light of theteachings contained herein. Examples of the products of the presentinvention and representative processes for their isolation, use, andmanufacture appear below.

The following examples are offered to more fully illustrate theinvention, but are not to be construed as limiting the scope thereof.

Example 1 Synthesis of3-(5-(methylthio)-1,3,4-thiadiazol-2-ylthio)pyrazine-2-carbonitrile

To a solution of 5-(methylthio)-1,3,4-thiadiazole-2-thiol (821 mg, 5.0mmol) in DMF and benzene (10 ml, 1/1) was added NaH (60% dispersion inmineral oil, 220 mg, 5.5 mmol) slowly at 0° C. under nitrogenatmosphere. The resulting suspension was stirred at 0° C. for 15 minutesand then to the mixture was added 3-chloropyrazine-2-carbonitrile (698mg, 5.0 mmol). The reaction was stirred at 80° C. for 4 hr. The reactionwas then cooled to room temperature and quenched with saturated NH₄Clsolution and extracted with EtOAc. The combined organic layers werewashed with water, brine and dried over MgSO₄, filtered and the filtratewas concentrated. The residue was purified by chromatography on silicagel to give the title compound as a white solid.

¹H-NMR (CDCl₃) δ 2.83 (s, 3H), 8.54 (d, J=1.8 Hz, 1H), 8.58 (d, J=1.8Hz, 1H). Mass Spectrum (ESI) m/e=268 (M⁺+1).

Example 2 Synthesis of3-(5-(ethylthio)-1,3,4-thiadiazol-2-ylthio)pyrazine-2-carbonitrile

The title compound was prepared according to the procedure described inExample 1 by using 5-(ethylthio)-1,3,4-thiadiazole-2-thiol (592 mg, 3.33mmol), NaH (60% dispersion in mineral oil, 146 mg, 3.66 mmol), and3-chloropyrazine-2-carbonitrile (422 mg, 3.02 mmol) in DMF and benzene(8 ml, 1/1) by stirring at 90° C. under nitrogen atmosphere overnight.

¹H-NMR (CDCl₃) δ 1.56 (t, J=5.6 Hz, 3H), 3.41 (q, J=5.6 Hz, 2H), 8.47(d, J=1.8 Hz, 1H), 8.52 (d, J=1.8 Hz, 1H). Mass Spectrum (ESI) m/e=(ESI)m/e=282 (M⁺+1).

Example 3 Synthesis of3-(5-(allylthio)-1,3,4-thiadiazol-2-ylthio)pyrazine-2-carbonitrile

The title compound was prepared according to the procedure described inExample 1 by using 5-(allylthio)-1,3,4-thiadiazole-2-thiol (460 mg, 2.42mmol), NaH (60% dispersion in mineral oil, 107 mg, 2.66 mmol), and3-chloropyrazine-2-carbonitrile (338 mg, 2.42 mmol) in DMF and benzene(8 ml, 1/1) by stirring at 85° C. under nitrogen atmosphere overnight.

¹H-NMR (CDCl₃) δ 4.02 (d, J=10.4 Hz, 2H), 5.25 (d, J=10.4 Hz, 1H), 5.39(d, J=15.6 Hz, 1H), 5.95-6.05 (m, 1H), 8.55 (d, J=1.8 Hz, 1H), 8.59 (d,J=1.8 Hz, 1H). Mass Spectrum (ESI) m/e=(ESI) m/e=294 (M⁺+1).

Example 4 Synthesis of3-(5-(propylthio)-1,3,4-thiadiazol-2-ylthio)pyrazine-2-carbonitrile

To a solution of 1,3,4-thiadiazole-2,5-dithiol (601 mg, 4.00 mmol) inDMF and benzene (6 ml, 1/1) was added NaH (60% dispersion in mineraloil, 176 mg, 4.40 mmol) slowly at 0° C. under nitrogen atmosphere. Theresulting suspension was stirred at 0° C. for 15 minutes and then to themixture was added bromopropane (492 mg, 2.00 mmol). The reaction wasstirred at rt for 1 hr. To the reaction was added NaH (60% dispersion inmineral oil, 176 mg, 4.40 mmol) slowly at 0° C. and stirred for 15minutes after addition. Then, 3-chloropyrazine-2-carbonitrile (557 mg,4.00 mmol) was added to the mixture and the reaction was stirred at 50°C. under N₂ overnight. The reaction was then cooled to room temperatureand quenched with saturated NH₄Cl solution and extracted with EtOAc. Thecombined organic layers were washed with water, brine and dried overMgSO4. Removal of solvent gave the crude product which was purified bychromatography to give the title compound as an off-white solid.

¹H-NMR (CDCl₃) δ 1.08 (t, J=5.5 Hz, 3H), 1.8-1.9 (m, 2H), 3.37 (q, J=5.5Hz, 2H), 8.52 (d, J=1.8 Hz, 1H), 8.58 (d, J=1.8 Hz, 1H). Mass Spectrum(ESI) m/e=296 (M⁺+1).

Example 5 Synthesis of3-(5-(butylthio)-1,3,4-thiadiazol-2-ylthio)pyrazine-2-carbonitrile

The title compound was prepared according to the procedure described inExample 4 by using 1,3,4-thiadiazole-2,5-dithiol (300 mg, 2.00 mmol),NaH (60% dispersion in mineral oil, 88 mg, 2.20 mmol), 1-iodobutane(0.263 ml, 2.30 mmol), NaH (60% dispersion in mineral oil, 88 mg, 2.20mmol), and 3-chloropyrazine-2-carbonitrile (280 mg, 2.00 mmol) in DMFand benzene (8 ml, 1/1) by stirring at room temperature under nitrogenatmosphere overnight.

¹H-NMR (CDCl₃) δ 0.98 (t, J=7.6 Hz, 3H), 1.44-1.58 (m, 2H), 1.78-1.85(m, 2H), 3.41 (t, J=7.6 Hz, 2H), 8.53 (d, J=2.4 Hz, 1H), 8.57 (d, J=2.4Hz, 1H). Mass Spectrum (ESI) m/e=310 (M⁺+1).

Example 6 Synthesis of3-(5-(isobutylthio)-1,3,4-thiadiazol-2-ylthio)pyrazine-2-carbonitrile

The title compound was prepared according to the procedure described inExample 4 by using 1,3,4-thiadiazole-2,5-dithiol (300 mg, 2.00 mmol),NaH (60% dispersion in mineral oil, 88 mg, 2.20 mmol),1-bromo-2-methylpropane (0.25 ml, 2.30 mmol), NaH (60% dispersion inmineral oil, 88 mg, 2.20 mmol), and 3-chloropyrazine-2-carbonitrile (280mg, 2.00 mmol) in DMF and benzene (8 ml, 1/1) by stirring at roomtemperature under nitrogen atmosphere overnight.

¹H-NMR (CDCl₃) δ 1.08 (dd, J=1.2, 6.8 Hz, 6H), 2.05-2.15 (m, 1H), 3.30(t, J=5.6 Hz, 2H), 8.53 (d, J=2.4 Hz, 1H), 8.57 (d, J=2.4 Hz, 1H). MassSpectrum (ESI) m/e=310 (M⁺+1).

Example 7 Synthesis of3-(5-(pentylthio)-1,3,4-thiadiazol-2-ylthio)pyrazine-2-carbonitrile

The title compound was prepared according to Example 4 by using1,3,4-thiadiazole-2,5-dithiol (300 mg, 2.00 mmol), NaH (60% dispersionin mineral oil, 88 mg, 2.20 mmol), 1-iodopentane (0.30 ml, 2.30 mmol),NaH (60% dispersion in mineral oil, 88 mg, 2.20 mmol), and3-chloropyrazine-2-carbonitrile (280 mg, 2.00 mmol) in DMF and benzene(8 ml, 1/1) by stirring at room temperature under nitrogen atmosphereovernight.

¹H-NMR (CDCl₃) δ 0.92 (t, J=7.6 Hz, 3H), 1.21-1.31 (m, 2H), 1.32-1.49(m, 4H), 1.81-1.89 (m, 2H), 3.38 (t, J=6.8 Hz, 2H), 8.53 (d, J=2.4 Hz,1H), 8.58 (d, J=2.4 Hz, 1H).

Mass Spectrum (ESI) m/e=324 (M+1).

Example 8 Synthesis of3-(5-(dodecylthio)-1,3,4-thiadiazol-2-ylthio)pyrazine-2-carbonitrile

The title compound was prepared according to Example 1 by using5-(dodecylthio)-1,3,4-thiadiazole-2-thiol (319 mg, 1.00 mmol), NaH (60%dispersion in mineral oil, 44 mg, 1.10 mmol), and3-chloropyrazine-2-carbonitrile (140 mg, 1.00 mmol) in DMF and benzene(6 ml, 1/1) by stirring at room temperature under nitrogen atmosphereovernight.

¹H-NMR (CDCl₃) δ 0.89 (t, J=5.4, 3H), 1.2-1.4 (m, 18H), 1.4-1.5 (m, 2H),1.8-1.9 (m, 2H), 3.39 (t, J=5.4, 2H), 8.54 (d, J=1.8 Hz, 1H), 8.58 (d,J=1.8 Hz, 1H). Mass Spectrum (ESI) m/e=422 (M⁺+1).

Example 9 Synthesis of3-(5-(benzylthio)-1,3,4-thiadiazol-2-ylthio)pyrazine-2-carbonitrile

To a solution of 5-(benzylthio)-1,3,4-thiadiazole-2-thiol (240 mg, 1.00mmol) in DMF and benzene (4 ml, 1/1) was added NaH (60% dispersion inmineral oil, 44 mg, 1.10 mmol) slowly at 0° C. under a nitrogenatmosphere. The resulting suspension was stirred at 0° C. for 15 minutesand then to the mixture was added 3-chloropyrazine-2-carbonitrile (140mg, 1.00 mmol). The reaction was stirred at room temperature for 2 hr.The reaction was quenched with saturated NH₄Cl solution and extractedwith EtOAc. The combined organic layers were washed with water, brineand dried over MgSO₄. Removal of the solvent gave the crude productwhich was purified by chromatography to give the title compound asoff-white solid.

¹H-NMR (CDCl₃) δ 4.63 (s, 2H), 7.20-7.43 (m, 5H), 8.54 (d, J=1.8 Hz,1H), 8.56 (d, J=1.8 Hz, 1H). Mass Spectrum (ESI) m/e=344 (M⁺+1).

Example 10 Synthesis of3-(5-mercapto-1,3,4-thiadiazol-2-ylthio)pyrazine-2-carbonitrile

The title compound was prepared according to the procedure described inExample 1 by using 1,3,4-thiadiazole-2,5-dithiol (945 mg, 6.05 mmol),NaH (60% dispersion in mineral oil, 264 mg, 6.60 mmol), and3-chloropyrazine-2-carbonitrile (840 mg, 6.00 mmol) in DMF and benzene(10 ml, 1/1) by stirring at 50° C. under nitrogen atmosphere for 4 hr.

¹H-NMR (DMSO) δ 8.77 (s, 1H), 8.88 (s, 1H). Mass Spectrum (ESI) m/e=254(M−⁺+1).

Example 11 Synthesis of3-(5-(isopropylthio)-4-methyl-4H-1,2,4-triazol-3-ylthio)pyrazine-2-carbonitrile

The title compound was prepared according to Example 1 by using5-(isopropylthio)-4-methyl-4H-1,2,4-triazole-3-thiol (379 mg, 2.00mmol), NaH (60% dispersion in mineral oil, 88 mg, 2.20 mmol), and3-chloropyrazine-2-carbonitrile (280 mg, 2.00 mmol) in DMF and benzene(6 ml, 1/1) by stirring at room temperature under nitrogen atmospherefor 2 hr.

¹H-NMR (CDCl₃) δ 1.49 (d, J=5.4 Hz, 6H), 3.57 (s, 3H), 4.01 (m, 1H),8.43 (d, J=1.8 Hz, 1H), 8.48 (d, J=1.8 Hz, 1H). Mass Spectrum (ESI)m/e=293 (M⁺+1).

Example 12 Synthesis of3-(5-(methylthio)-1,2,4-thiadiazol-3-ylthio)pyrazine-2-carbonitrile

The title compound was prepared according to Example 1 by using5-(methylthio)-1,2,4-thiadiazole-3-thiol (328 mg, 2.00 mmol), NaH (60%dispersion in mineral oil, 88 mg, 2.20 mmol), and3-chloropyrazine-2-carbonitrile (280 mg, 2.00 mmol) in DMF and benzene(6 ml, 1/1) by stirring at room temperature under nitrogen atmospherefor 2 hr.

¹H-NMR (CDCl₃) δ 2.72 (s, 3H), 8.66 (d, J=1.8 Hz, 1H), 8.82 (d, J=1.8Hz, 1H). Mass Spectrum (ESI) m/e=268 (M⁺+1).

Example 13 Synthesis of3-(5-methyl-1,3,4-thiadiazol-2-ylthio)pyrazine-2-carbonitrile

The title compound was prepared according to Example 1 by using5-methyl-1,3,4-thiadiazole-2-thiol (264 mg, 2.00 mmol), NaH (60%dispersion in mineral oil, 88 mg, 2.20 mmol), and3-chloropyrazine-2-carbonitrile (280 mg, 2.00 mmol) in DMF and benzene(6 ml, 1/1) by stirring at room temperature under nitrogen atmospherefor 6 hr.

¹H-NMR (CDCl₃) δ 2.86 (s, 3H), 8.52 (d, J=1.8 Hz, 1H), 8.55 (d, J=1.8Hz, 1H). Mass Spectrum (ESI) m/e=236 (M⁺+1).

Example 14 Synthesis of3-(5-butyl-1,3,4-thiadiazol-2-ylthio)pyrazine-2-carbonitrile

The title compound was prepared according to Example 1 by using5-butyl-1,3,4-thiadiazole-2-thiol (260 mg, 1.49 mmol), NaH (60%dispersion in mineral oil, 66 mg, 1.65 mmol), and3-chloropyrazine-2-carbonitrile (210 mg, 1.50 mmol) in DMF and benzene(4 ml, 1/1) by stirring at room temperature under nitrogen atmospherefor 3 hr.

¹H-NMR (CDCl₃) δ 1.01 (t, J=7.6 Hz, 3H), 1.42-1.49 (m, 2H), 1.81-1.91(m, 2H), 3.19 (t, J=8.0 Hz, 2H), 8.53 (d, J=1.8 Hz, 1H), 8.57 (d, J=1.8Hz, 1H). Mass Spectrum (ESI) m/e=278 (M⁺+1).

Example 15 Synthesis of3-(4-methyl-4H-1,2,4-triazol-3-ylthio)pyrazine-2-carbonitrile

The title compound was prepared according to Example 1 by using4-methyl-4H-1,2,4-triazole-3-thiol (230.5 mg, 2.00 mmol), NaH (60%dispersion in mineral oil, 88 mg, 2.20 mmol), and3-chloropyrazine-2-carbonitrile (280 mg, 2.00 mmol) in DMF and benzene(6 ml, 1/1) by stirring at room temperature under nitrogen atmospherefor 4 hr.

¹H-NMR (CDCl₃) δ 3.75 (s, 3H), 8.43 (d, J=1.8 Hz, 1H), 8.48 (d, J=1.8Hz, 1H). Mass Spectrum (ESI) m/e=219 (M⁺+1).

Example 16 Synthesis of3-(1-methyl-1H-imidazol-2-ylthio)pyrazine-2-carbonitrile

The title compound was prepared according to Example 1 by using1-methyl-1H-imidazole-2-thiol (228 mg, 2.00 mmol), NaH (60% dispersionin mineral oil, 88 mg, 2.20 mmol), and 3-chloropyrazine-2-carbonitrile(280 mg, 2.00 mmol) in DMF and benzene (6 ml, 1/1) by stirring at roomtemperature under nitrogen atmosphere for 4 hr.

¹H-NMR (CDCl₃) δ 3.74 (s, 3H), 7.22 (d, J=1.6 Hz, 1H), 7.28 (d, J=1.6Hz, 1H), 8.40 (d, J=1.8 Hz, 1H), 8.44 (d, J=1.8 Hz, 1H). Mass Spectrum(ESI) m/e=218 (M⁺+1).

Example 17 Synthesis of2-chloro-3-(5-(methylthio)-1,3,4-thiadiazol-2-ylthio)pyrazine

The title compound was prepared according the procedure described inExample 1 by using 5-(methylthio)-1,3,4-thiadiazole-2-thiol (1.64 g,10.00 mmol), NaH (60% dispersion in mineral oil, 445 mg, 11.00 mmol),and 2,3-dichloropyrazine (1.04 ml, 10.00 mmol) in DMF and benzene (12ml, 1/1) by stirring at 50° C. for 20 hr and then at 110° C. for 4 hrunder nitrogen atmosphere.

¹H-NMR (CDCl₃) δ 2.85 (s, 3H), 8.22 (d, J=1.8 Hz, 1H), 8.35 (d, J=1.8Hz, 1H).

Mass Spectrum (ESI) m/e=277 (M⁺+1).

Example 18 Isolation of LCAT

LCAT was isolated from culture media of CHO cells that were stablytransfected with human LCAT cDNA. LCAT coding sequence was tagged withFLAG and HIS tages at the C-terminus. Purification of tagged recombinanthuman LCAT protein was performed according to the standard protocolusing Ni-NTA agarose beads. Briefly, CHO cultured media were incubatedwith Ni-NTA column at pH 8.0. The unbound proteins were washed fromresin complex with 20 mM imidazole. The His-tagged protein was elutedwith 250 mM imidazole and dialysed against 1×PBS containing 50 μM EDTA.

Example 19 LCAT Activity Assays

Activity of the modified LCAT of the invention and activities ofcompounds of the invention were determined by measuring change of therate of conversion of ³H-labeled cholesterol to cholesteryl ester (CE).In the plasma LCAT activity assay, human plasma samples wereequilibrated with a trace amount of radiolabeled cholesterol at 4° C.and the rate of cholesterol esterification was measured by TLC analysisafter incubation at 37° C. (Dobiasova, supra). EC₅₀ represents acompound concentration achieving 50% of maximal activation ofLCAT-mediated cholesterol esterification.

For measuring compound activity using apoAI-liposome assay format,full-length human LCAT cDNA was isolated from normal human liver cDNAlibrary (BioChain, Hayward, Calif.) with standard protocol and clonedinto a pCMV-Flag vector (See Example 18). Recombinant LCAT was expressedin CHO cells and the enzyme secreted from stably transfected cells washarvested in serum-free culture medium. Recombinant LCAT identity wasconfirmed with anti-human LCAT and anti-Flag antibodies. The activity ofrecombinant LCAT enzyme was determined using apoAI-liposome substratesprepared by the standard cholate-dialysis procedure (Chen et al. (1982)J. Lipid Res. 23: 680-691. The initial mixture contained eggPC/³H-unesterfied cholesterol/human apoAI (molar ratio of 250:12.5:0.8).After dialysis the proteoliposomes were incorporated with recombinantLCAT protein. LCAT activity was determined by measuring the conversionof radiolabeled cholesterol to cholesteryl ester and expressed in nmolCE/mL per hour.

Example 20 LCAT Stability Measurements

LCAT enzyme stability was measured using the standard ELISA protocol.Briefly, LCAT protein molecules of the plasma samples were captured ontothe ELISA plate with an anti-LCAT antibody which was pre-coated to theplate. After a careful wash to remove the unbound molecules, the LCATprotein was detected by using a second anti-LCAT antibody. TheLCAT-antibody immunocomplexes were detected and quantified by using HRPdetection system. Purified recombinant LCAT protein were used asstandard and measured under the same experiment conditions (Kobori etal. (2002) J Lipid Res 43: 325-334).

Example 21 Assessment of the In Vivo Effects of the Modified LCAT

Rodents used for assessing the in vivo effects of the compounds of theinvention include BALB/c mice, CD1 mice, and Syrian hamsters of wildtype fed with normal chew. These animals were treated with eithervehicle or Compound A(3-(5-ethylthio)-1,3,4-thiadiazol-2-ylthio)pyrazine-2-carbonitrile) byIP injection. At time points (see Figures) after compoundadministration, blood samples were collected, plasma was immediatelyseparated, and plasma levels of lipids, lipoprotein and LCAT activitywere determined.

Results presented in FIG. 2A confirm activity and specificity of thecompounds of the invention on LCAT enzyme. This experiment studied theeffect of Compound A in LCAT-deficient plasma samples obtained from lcatmutant mice. Ng et al. (1997) J. Biol. Chem. 272:1 5777-81. No LCATactivity was detected in LCAT-deficient (lcat −/−) plasma samples in thepresence or absence of Compound A, indicating that the observedinteraction of Compound A with LCAT is highly specific.

Results summarized in FIG. 2B illustrate the mechanism of action ofcompounds of the invention on LCAT enzyme. Two loss-of-function LCATmutants, H377A and S181A were generated, in which the critical catalytictriad of the enzyme was destroyed as described in Francone et al. (1991)Biochemistry 30: 10074-77; Peelman et al. (1998) Protein Sci. 7:587-599; and Peelman et al. (2000) Curr. Opin. Lipidol. 11: 155-160. Noactivity was observed on either nonfunctional enzyme with or withoutCompound A (FIG. 2B). To test a hypothesis whether the mechanism of LCATactivation might involve the reaction of the molecule with a free thiolgroup in the enzyme, two other mutations in LCAT polypeptide targetingcysteine residues C31 and C184 were made. Neither of these two mutationssignificantly altered basal LCAT activity of the recombinant proteins.However, they exhibited distinct responses to Compound A. While theC184A mutant was able to respond to the treatment in a dose dependentmanner comparable to that of the wild type enzyme, the C31A mutantfailed to be activated by Compound A. This observation is consistentwith the hypothesis that compounds of the invention bind irreversibly toLCAT at the amino acid residue 31 as measured in biochemical assays.

FIG. 3 summarizes data showing that compounds of the invention increaseLCAT enzyme activity in a dose dependent manner in BALB/c mice. Briefly,BALB/c mice (male, 7 weeks old) were treated with either vehicle orCompound A by intra-peritoneal (IP) in the indicated doses. Animals werefed normal chow diet. Compounds were solubilized in DEPG vehicle(containing 20% dimethyl acetamide, 10% ethanol, 50% polylene glycol)and administered to the animals. At the indicated time points, bloodsamples were taken from animals and plasma was separated immediately. Analiquot of plasma sample was labeled with ³H-cholesterol for the LCATactivity assay. Each data point represents the mean of samples from twoindividual animals. The remaining samples were used for plasma lipid andlipoprotein analyses.

Results presented in FIG. 4 demonstrate that treatment with thecompounds of the invention increases HDL cholesterol levels in CD1 mice.CD1 male three month old mice were treated with either vehicle (control)or with Compound A by IP injection (20 mg/kg, one dose per day, 4 days,n=8). Plasma samples were collected and HDL cholesterol concentrationswere determined standard reagents and assay protocol using clinicalanalyzer (Infinity).

FIG. 5 illustrates the time course of LCAT activation and the levels ofHDL in mice following a single doze of Compound A. Male three month oldCD1 mice were given Compound A (20 mg/kg) by an IP injection. At eachindicated time point, a group of animals (n=4) was sacrificed, bloodsamples were collected and plasma separated for measurement of LCATactivity (diamonds) and HDL (circles). Each data point represents themean of measurement from four individual animals per treatment group.

Results presented in FIG. 6 demonstrate that treatment with the compoundof the invention increases HDL levels and decreases apoB-containinglipoprotein in vivo. Hamsters (Syrian, male, 12 weeks old, n=6 pergroup) were treated with either vehicle (control) or Compound A via IPadministration (20 mg/kg, one dose per day, 4 days). Plasma samples werecollected and concentrations of total cholesterol (TC), FIG. 6B, and HDLcholesterol (FIG. 6A) were measured. LpB cholesterol contents wereobtained by subtracting HDL from TC (FIG. 6C).

FIG. 7 provides elution profiles indicating that treatment with thecompounds of the invention increases HDL-Ch levels, increases HDLparticle size, and decrease TG levels in the VLDL fraction in vivo.Hamsters (n=6) were treated with either vehicle (control), or Compound Avia IP administration (20 mg/kg/day, 4 days). Plasma samples were pooledwithin each treatment group, and separated by FPLC using two seriallyconnected Superose 6 columns (Pharmacia Biotech Inc.). Cholesterol (FIG.7A) and triglyceride (FIG. 7B) levels were determined in 0.5 mlfractions.

TABLE 1 HDL particle size profiles HDL subclass Control T865 2a 12 39 2b58 56 3a 28 3 3b 1 1 3c 1 1

Table 1 summarizes HDL particle size profiles as determined by GradientGel Electrophoresis (GGE) indicating an increase of HDL particle size invivo after treating with the compounds of the invention. Hamsters weretreated with either vehicle or Compound A as described above (20mg/kg/day, 4 days, n=6). Plasma samples were collected and pooled foreach group. An aliquot of pooled plasma was analyzed using GGE asdescribed in Blanche et al. (1981) BBA 665: 408-419.

All publications, patents and patent applications cited in thisspecification are herein incorporated by reference as if each individualpublication or patent application were specifically and individuallyindicated to be incorporated by reference. Although the foregoinginvention has been described in some detail by way of illustration andexample for purposes of clarity of understanding, it will be readilyapparent to those of ordinary skill in the art in light of the teachingsof this invention that certain changes and modifications may be madethereto without departing from the spirit or scope of the appendedclaims.

What is claimed is:
 1. A method for treating atherosclerosis in asubject comprising administering to the subject a therapeuticallyeffective amount of a compound of Formula I

wherein X, Y and Z are independently selected from the group consistingof —N═, —S—, —CH═ and

provided that at least two of X, Y and Z are not —S—, and provided thatno more than one of X, Y and Z is —CH═; L is —S—, —S(O)—, or —S(O)₂—; R¹is selected from the group consisting of CN, COOR⁵, SO₂R⁶ and halogen;R² is selected from the group consisting of H, optionally substitutedC₁-C₁₂ alkyl, optionally substituted C₁-C₁₂ alkenyl, optionallysubstituted C₁-C₈ alkynyl, optionally substituted aryl, and optionallysubstituted heteroaryl, and SR³, wherein the substituents are selectedfrom the group consisting of C₁-C₄ alkyl, NH₂, halo and CN; and whereinR³ is selected from the group consisting of H, optionally substitutedC₁-C₁₂ alkyl, optionally substituted C₁-C₈ alkenyl, optionallysubstituted C₁-C₈ alkynyl, optionally substituted aryl and optionallysubstituted heteroaryl, wherein the substituents are selected from thegroup consisting of NH₂, halo and CN; R⁴ is H or C₁-C₈ alkyl; R⁵ and R⁶are each independently C₁-C₄ alkyl; or a pharmaceutically acceptablesalt thereof.
 2. The method of claim 1, wherein X and Y are each —N═. 3.The method of claim 2, wherein Z is —S—.
 4. The method of claim 1,wherein L is —S—.
 5. The method of claim 1, wherein R¹ is CN.
 6. Themethod of claim 1, wherein R² is SR³.
 7. The method of claim 1, whereinR³ is C₁-C₄ alkyl.
 8. The method of claim 7, wherein R³ is methyl. 9.The method of claim 1, wherein the compound is selected from the groupconsisting of3-(5-(Methylthio)-1,3,4-thiadiazol-2-ylthio)pyrazine-2-carbonitrile3-(5-(Ethylthio)-1,3,4-thiadiazol-2-ylthio)pyrazine-2-carbonitrile3-(5-(Allylthio)-1,3,4-thiadiazol-2-ylthio)pyrazine-2-carbonitrile3-(5-(Propylthio)-1,3,4-thiadiazol-2-ylthio)pyrazine-2-carbonitrile3-(5-(Butylthio)-1,3,4-thiadiazol-2-ylthio)pyrazine-2-carbonitrile3-(5-(Isobutylthio)-1,3,4-thiadiazol-2-ylthio)pyrazine-2-carbonitrile3-(5-(Pentylthio)-1,3,4-thiadiazol-2-ylthio)pyrazine-2-carbonitrile3-(5-(Dodecylthio)-1,3,4-thiadiazol-2-ylthio)pyrazine-2-carbonitrile3-(5-(Benzylthio)-1,3,4-thiadiazol-2-ylthio)pyrazine-2-carbonitrile3-(5-Mercapto-1,3,4-thiadiazol-2-ylthio)pyrazine-2-carbonitrile3-(5-(Isopropylthio)-4-methyl-4H-1,2,4-triazol-3-ylthio)pyrazine-2-carbonitrile3-(5-(Methylthio)-1,2,4-thiadiazol-3-ylthio)pyrazine-2-carbonitrile3-(5-Methyl-1,3,4-thiadiazol-2-ylthio)pyrazine-2-carbonitrile3-(5-Butyl-1,3,4-thiadiazol-2-ylthio)pyrazine-2-carbonitrile3-(4-Methyl-4H-1,2,4-triazol-3-ylthio)pyrazine-2-carbonitrile3-(1-Methyl-1H-imidazol-2-ylthio)pyrazine-2-carbonitrile, and2-Chloro-3-(5-(methylthio)-1,3,4-thiadiazol-2-ylthio)pyrazine or apharmaceutically acceptable salt thereof.
 10. A method for treatingatherosclerosis in a subject in need thereof, comprising administering atherapeutically effective amount of a modified LCAT comprising areplacement of the amino acid residue 31 by a cysteine residue, whereinthe cysteine residue is modified by replacing the thiol hydrogen with3-pyrazinyl-2-carbonitrile.
 11. The method of claim 10, wherein themodified LCAT is administered intravenously.
 12. The method of claim 11,wherein the modified LCAT is administered by bolus.
 13. A method fortreating an LCAT-mediated disease comprising administering to a subjectin need thereof an effective amount of a modified LCAT comprising areplacement of the amino acid residue 31 by a cysteine residue, whereinthe cysteine residue is modified by replacing the thiol hydrogen with3-pyrazinyl-2-carbonitrile.
 14. The method of claim 13, wherein theLCAT-mediated disease is selected from the group consisting ofatherosclerosis, thrombosis, coronary heart disease, high bloodpressure, LCAT deficiency syndrome, Alzheimer's disease, cornealopacity, metabolic syndrome, dyslipidemia, myocardial infartion, stroke,critical limb ischemia, angina.
 15. A method for increasing HDLcholesterol in a subject comprising administering to the subject apharmaceutical composition comprising a therapeutically effective amountof a modified LCAT comprising a replacement of the amino acid residue 31by a cysteine residue, wherein the cysteine residue is modified byreplacing the thiol hydrogen with 3-pyrazinyl-2-carbonitrile, and apharmaceutically acceptable carrier or excipient.
 16. A method forpreventing accumulation of cholesterol in a subject comprisingadministering to the subject a pharmaceutical composition comprising atherapeutically effective amount of a modified LCAT comprising areplacement of the amino acid residue 31 by a cysteine residue, whereinthe cysteine residue is modified by replacing the thiol hydrogen with3-pyrazinyl-2-carbonitrile, and a pharmaceutically acceptable carrier orexcipient.
 17. A method for treating atherosclerosis in a subject inneed thereof, comprising administering a pharmaceutical compositioncomprising a therapeutically effective amount of a modified LCATcomprising a replacement of the amino acid residue 31 by a cysteineresidue, wherein the cysteine residue is modified by replacing the thiolhydrogen with 3-pyrazinyl-2-carbonitrile and a pharmaceuticallyacceptable carrier or excipient.
 18. A pharmaceutical compositioncomprising a modified LCAT comprising a replacement of the amino acidresidue 31 by a cysteine residue, wherein the cysteine residue ismodified by replacing the thiol hydrogen with3-pyrazinyl-2-carbonitrile, and a pharmaceutically acceptable carrier.19. The method of claim 1, wherein the subject is mammal.
 20. The methodof claim 1, wherein the subject is human.